Bis-sulfhydryl macrocyclization systems

ABSTRACT

The present invention provides novel peptidomimetic macrocycles and methods for their preparation and use, as well as amino acid analogs and macrocycle-forming linkers, and kits useful in their production.

CROSS-REFERENCE

This application is a continuation application of U.S. application Ser.No. 11/957,325, filed Dec. 14, 2007, which claims benefit to U.S.Provisional Application Ser. No. 60/874,819, filed Dec. 14, 2006, all ofwhich is incorporated herein by reference in its entirety and to whichapplication we claim priority under 35 USC §120.

BACKGROUND OF THE INVENTION

Peptides are becoming increasingly important in drug discovery.Unmodified peptides often suffer from poor metabolic stability, poorcell penetrability, and promiscuous binding due to conformationalflexibility. To improve these properties, researchers have generatedcyclic peptides and peptidomimetics by a variety of methods, includingdisulfide bond formation, amide bond formation, and carbon-carbon bondformation (Jackson et al. (1991), J. Am. Chem. Soc. 113:9391-9392;Phelan et al. (1997), J. Am. Chem. Soc. 119:455-460; Taylor (2002),Biopolymers 66: 49-75; Brunel et al. (2005), Chem. Commun.(20):2552-2554; Hiroshige et al. (1995), J. Am. Chem. Soc. 117:11590-11591; Blackwell et al. (1998), Angew. Chem. Int. Ed.37:3281-3284; Schafmeister et al. (2000), J. Am. Chem. Soc.122:5891-5892). Limitations of these methods include poor metabolicstability (disulfide and amide bonds), poor cell penetrability(disulfide and amide bonds), and the use of potentially toxic metals(carbon-carbon bonds).

SUMMARY OF THE INVENTION

The present invention provides novel peptidomimetic macrocycles andmethods for their preparation and use. In general, the synthesis ofthese peptidomimetic macrocycles involves (1) synthesizing a precursorpeptide containing two free —SH moieties; and (2) contacting theprecursor peptide with a bis-alkylating reagent to yield a novelpeptidomimetic macrocycle. This general method permits the covalentlinkage of at least two free thiolate moieties in a precursor peptide toyield novel compounds that exhibit improved biological properties suchas structural stability, affinity for a target, resistance toproteolytic degradation and cell penetrance. In addition, this generalmethod permits the rapid and selective incorporation of a broaddiversity of moieties into the peptidomimetic macrocycle to permit thegeneration of a library of related macrocycles. This general method alsopermits the facile incorporation of labels (e.g., radioisotopes,chemiluminescent or fluorescent labels) or therapeutic agents.

Thus, in one aspect, the invention provides a peptidomimetic macrocycleof Formula (I):

wherein:each A, C, D, and E is independently a natural or non-natural aminoacid;B is a natural or non-natural amino acid, amino acid analog

[—NH-L₄-CO—], [—NH-L₄-SO₂—], or [—NH-L₄-]:R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-:R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, unsubstituted or substituted with R₅;L₁, L₂, L₃ and L₄ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene,heterocycloalkylene, cycloarylene, heterocycloarylene or [—R₄—K—R₄-]n,each being unsubstituted or substituted with R₅:K is O, S, SO, SO₂, CO, CO₂, or CONR₃;each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene:each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,unsubstituted or substituted with R₅, or part of a cyclic structure witha D residue;R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,unsubstituted or substituted with R₅, or part of a cyclic structure withan E residue;v is an integer from 1-1000;w is an integer from 1-1000;x is an integer from 0-10;y is an integer from 0-10;z is an integer from 0-10;n is an integer from 1-5; andx+y+z is at least 3.

In some embodiments the peptidomimetic macrocycle comprises an α-helixand R₈ is —H. In some embodiments, at least one of R₁ and R₂ is alkyl,alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl,or heterocycloalkyl, unsubstituted or substituted with halo-.Alternatively, both R₁ and R₂ are independently alkyl, alkenyl, alkynyl,arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, orheterocycloalkyl, unsubstituted or substituted with halo-. In otherembodiments, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-, or both R₁ and R₂ are independently alkyl,unsubstituted or substituted with halo-. In yet other embodiments, atleast one of R₁ and R₂ is methyl, or both R₁ and R₂ are methyl.

In some embodiments, at least one of D and E is a natural or unnaturalamino acid substituted with a high molecular weight lipid orhydrocarbon. In other embodiments, at least one of D and E is attachedto an additional macrocycle-forming linker of the formula[-L₁-S-L₂-S-L₃-].

In some cases, a secondary structure of the peptidomimetic macrocycle ismore stable than a corresponding secondary structure of a correspondingnon-macrocyclic polypeptide. In some embodiments, the peptidomimeticmacrocycle of the invention also comprises an α-helix. Such an α-helix,for example, comprises from 1 turn to 5 turns. Such an α-helix is, forexample, more stable than an α-helix of a corresponding non-macrocyclicpolypeptide. In some embodiments, [-L₁-S-L₂-S-L₃-] spans from 1 turn to5 turns of the α-helix, such as approximately 1, 2, 3, 4 or 5 turns ofthe α-helix. For example, [-L₁-S-L₂-S-L₃-] spans approximately 1 turn ofthe α-helix. Exemplary lengths of [-L₁-S-L₂-S-L₃-] are about 5 Å toabout 9 Å per turn of the α-helix. In some embodiments, the length of[-L₁-S-L₂-S-L₃-] is approximately equal to the length of from about 5carbon-carbon bonds to about 13 carbon-carbon bonds, or from about 7carbon-carbon bonds to about 10 carbon-carbon bonds. In otherembodiments, the macrocycle of the invention comprises a ring of about17 atoms to 25 atoms.

In yet other embodiments, the peptidomimetic macrocycle of the inventioncomprises an α-helix which comprises about 2 turns. For example, thelength of [-L₁-S-L₂-S-L₃-] is approximately equal to the length of fromabout 8 carbon-carbon bonds to about 16 carbon-carbon bonds, or fromabout 10 carbon-carbon bonds to about 13 carbon-carbon bonds. In otherembodiments, the macrocycle of the invention comprises a ring of about29 atoms to about 37 atoms.

The present invention also provides a compound of Formula IIa:

wherein:R₁ is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,heteroalkyl, or heterocycloalkyl;L₁ is independently alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene or[—R₄—K—R₄-]n, unsubstituted or substituted with R₅;K is O, S, SO, SO₂, CO, CO₂ or CONR₃:R₄ is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,heterocycloalkylene, arylene, or heteroarylene;R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂₆,—CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent;R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heteroalkylalkyl, heterocyclyalkyl, a fluorescentmoiety, a radioisotope or a therapeutic agent;R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl,heteroalkylalkyl, or heterocyclyalkyl:R₉ and R₁₀ are independently —H or a protecting group suitable forpeptide synthesis;n is an integer from 1 to 5;

Q is S; and

P is —H, -trityl, p-methoxytrityl, —S t-butyl, or any other protectinggroup suitable for peptide synthesis: or Q and P when taken togetherform a moiety capable of undergoing chemical transformation into an —SHgroup.

In some embodiments, R₁ is alkyl, unsubstituted or substituted withhalo-. In other embodiments, R₁ is unsubstituted alkyl. In yet otherembodiments. R₁ is methyl. In still other embodiments, at least one ofR₉ and R₁₀ is a protected group suitable for peptide synthesis.

The present invention also provides a kit comprising a) a compound ofFormulas IIa and a compound of Formula IIb:

wherein:R₁ is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, orheterocycloalkyl, unsubstituted or substituted with halo-:R₂ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,or heterocycloalkyl, unsubstituted or substituted with halo-;L₁ and L₃ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, orheterocycloarylene or [—R₄—K—R₄-]n, each being unsubstituted orsubstituted with R₅;K is O, S, SO, SO₂, CO, CO₂, or CONR₃;R₄ is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,heterocycloalkylene, arylene, or heteroarylene;each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, —R₆, a fluorescent moiety, a radioisotope, or atherapeutic agent;each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heteroalkylalkyl, heterocyclyalkyl, a fluorescentmoiety, a radioisotope, or a therapeutic agent;R₇ and R₈; are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heteroalkylalkyl, or heterocyclyalkyl;R₉ and R₁₀ are each independently H or any protecting group suitable forliquid or solid phase peptide synthesis;

Q is S;

P is —H, -trityl, p-methoxytrityl, —S t-butyl, or any other protectinggroup suitable for liquid or solid phase peptide synthesis; or Q and Pwhen taken together form a moiety capable of undergoing chemicaltransformation into an —SH group; n is an integer from 1 to 5;and b) a macrocycle-forming linker of the structure:

X-L₂-Y

wherein L₂ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or[—R₁₁—K—R₁₁-]n, each being unsubstituted or substituted with R₁₂;each R₁₁ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;each R₁₂ is independently halogen, alkyl, —OR₁₃, —N(R₆)₁₃, —SR₁₃,—SOR₁₃, —SO₂R₁₃, —CO₂R₁₃, —R₁₃, a fluorescent moiety, a radioisotope, ora therapeutic agent;each R₁₃ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heteroalkylalkyl, heterocyclyalkyl, a fluorescentmoiety, a radioisotope, or a therapeutic agent; andX and Y are each independently a reactive group capable of reacting witha thiol group.

In some embodiments, R₂ is alkyl, alkenyl, alkynyl, arylalkylcycloalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted orsubstituted with halo. In specific such embodiments, R₁ and R₂ arealkyl. For example, R₁ and R₂ are methyl or trifluoromethyl.

A method for synthesizing a peptidomimetic macrocycle, the methodcomprising the step of contacting a peptidomimetic precursor of theFormula III:

with a compound formula X-L₂-Y,wherein v, w, x, y, z, A, B, C, D, E, R₁, R₂, R₇, R₈, L₁, L₂, and L₃ areas defined for the compound of formula I; andX and Y are each independently a reactive group capable of reacting witha thiol group;x+y+z is at least 3;and further wherein said contacting step results in a covalent linkagebeing formed between the two thiol groups in Formula III.

In some embodiments, performing a method of the invention results in theformation of a peptidomimetic macrocycle of Formula (I) as describedherein.

In certain embodiments, at least one of R₁ and R₂ is alkyl, alkenyl,alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, orheterocycloalkyl, unsubstituted or substituted with halo-.Alternatively, both R₁ and R₂ are independently alkyl, alkenyl, alkynyl,arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, orheterocycloalkyl, unsubstituted or substituted with halo-. In otherembodiments, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-, or both R₁ and R₂ are independently alkyl,unsubstituted or substituted with halo-. In yet other embodiments, atleast one of R₁ and R₂ is methyl, or both R₁ and R₂ are methyl.

In some embodiments, the peptidomimetic precursor is expressed in cells.The peptidomimetic precursor is also purified, in some embodiments,prior to the contacting step. The obtained peptidomimetic macrocycle is,in some instances, purified after the contacting step, and/or refoldedafter the contacting step.

The described method is, for example, performed in solution, or itperformed on a solid support. The contacting step is, in some cases,performed in the presence of a target macromolecule that binds to thepeptidomimetic precursor under conditions that favor said binding, or itis performed in the presence of a target macromolecule that bindspreferentially to the peptidomimetic precursor under conditions thatfavor said binding. In some embodiments, the described method is appliedto synthesize a library of peptidomimetic macrocycles.

In some embodiments, a peptidomimetic macrocycle prepared by the methodof the invention comprises an α-helix in aqueous solution. In otherembodiments, the peptidomimetic macrocycle exhibits increased α-helicalstructure in aqueous solution compared to a correspondingnon-macrocyclic polypeptide. In still other embodiments, thepeptidomimetic macrocycle exhibits increased thermal stability,increased biological activity, increased resistance to proteolyticdegradation, or increased ability to penetrate living cells compared toa corresponding non-macrocyclic polypeptide. In some embodiments, thetwo thiol moieties of the compound of Formula III are sidechains of anamino acid selected from the group consisting of L-cysteine, D-cysteine,α-methyl L-cysteine, and α-methyl D-cysteine. In certain embodiments ofthe method of the invention, x+y+z is 3, and A, B and C areindependently natural or non-natural amino acids.

The method described is, for example, performed in a solvent selectedfrom the group consisting of protic solvent, aqueous solvent, organicsolvent, and mixtures thereof. In some embodiments, the solvent is DMF,dichloroethane, NH₃, NH₃/MeOH, NH₃/DMF, or aqueous guanidinium-HCL. Insome embodiments, the solvent is also be a solvent that favors helixformation, such as water.

In some embodiments of the compounds and methods described herein, L₂ isan alkyl group. In other embodiments, X and Y are independently chosenhalogen groups such as Cl—, Br— or I—.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows a MALDI spectrum of a peptidomimetic macrocycle of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

Definitions

As used herein, the term “macrocycle” refers to a molecule having achemical structure including a ring or cycle formed by at least 9covalently bonded atoms.

As used herein, the term “peptidomimetic macrocycle” refers to acompound comprising a plurality of amino acid residues joined by aplurality of peptide bonds and at least one macrocycle-forming linkerwhich forms a macrocycle between the α carbon of one naturally-occurringamino acid residue or non-naturally-occurring amino acid residue oramino acid analog residue and the α carbon of anothernaturally-occurring amino acid residue or non-naturally-occurring aminoacid residue or amino acid analog residue. The peptidomimeticmacrocycles optionally include one or more non-peptide bonds between oneor more amino acid residues and/or amino acid analog residues, andoptionally include one or more non-naturally-occurring amino acidresidues or amino acid analog residues in addition to any which form themacrocycle.

As used herein, the term “stability” refers to the maintenance of adefined secondary structure in solution by a peptide or peptidomimeticmacrocycle of the invention as measured by circular dichroism, NMR oranother biophysical measure, or resistance to proteolytic degradation invitro or in vivo. Non-limiting examples of secondary structurescontemplated in this invention are α-helices, n-turns, and 3-pleatedsheets.

As used herein, the term “helical stability” refers to the maintenanceof a helical structure by a peptide or peptidomimetic macrocycle of theinvention as measured by circular dichroism. For example, in someembodiments, the peptidomimetic macrocycles of the invention exhibit atleast a 1.25, 1.5, 1.75 or 2-fold increase in α-helicity as determinedby circular dichroism compared to a corresponding non-macrocyclicpolypeptide.

The term “α-amino acid” or simply “amino acid” refers to a moleculecontaining both an amino group and a carboxyl group bound to a carbonwhich is designated the α-carbon. Suitable amino acids include, withoutlimitation, both the D- and L-isomers of the naturally-occurring aminoacids, as well as non-naturally occurring amino acids prepared byorganic synthesis or other metabolic routes. Unless the contextspecifically indicates otherwise, the term amino acid, as used herein,is intended to include amino acid analogs.

The term “naturally occurring amino acid” refers to any one of thetwenty amino acids commonly found in peptides synthesized in nature, andknown by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L,K, M, F, P, S, T, W, Y and V.

The term “amino acid analog” refers to a molecule which is structurallysimilar to an amino acid and which can be substituted for an amino acidin the formation of a peptide or peptidomimetic macrocycle. Amino acidanalogs include compounds which are structurally identical to an aminoacid, as defined herein, except for the inclusion of one or moreadditional methylene groups between the amino and carboxyl group (e.g.,α-amino β-carboxy acids), or for the substitution of the amino orcarboxy group by a similarly reactive group (e.g., substitution of theprimary amine with a secondary or tertiary amine, or substitution or thecarboxy group with an ester).

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of a polypeptide (e.g., a BH3 domain or thep53 MDM2 binding domain) without abolishing or substantially alteringits essential biological or biochemical activity (e.g., receptor bindingor activation). An “essential” amino acid residue is a residue that,when altered from the wild-type sequence of the polypeptide, results inabolishing or substantially abolishing the polypeptide's essentialbiological or biochemical activity.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., K, R, H), acidic side chains (e.g., D, E), unchargedpolar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains(e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V,I) and aromatic side chains (e.g., Y, F, W, H). Thus, a predictednonessential amino acid residue in a BH3 polypeptide, for example, ispreferably replaced with another amino acid residue from the same sidechain family.

The term “member” as used herein in conjunction with macrocycles ormacrocycle-forming linkers refers to the atoms that form or can form themacrocycle, and excludes substituent or side chain atoms. By analogy,cyclodecane, 1,2-difluoro-decane and 1,3-dimethyl cyclodecane are allconsidered ten-membered macrocycles as the hydrogen or fluorosubstituents or methyl side chains do not participate in forming themacrocycle.

The symbol “

” when used as part of a molecular structure refers to a single bond ora trans or cis double bond.

The term “amino acid side chain” refers to a moiety attached to theα-carbon in an amino acid. For example, the amino acid side chain foralanine is methyl, the amino acid side chain for phenylalanine isphenylmethyl, the amino acid side chain for cysteine is thiomethyl, theamino acid side chain for aspartate is carboxymethyl, the amino acidside chain for tyrosine is 4-hydroxyphenylmethyl, etc. Othernon-naturally occurring amino acid side chains are also included, forexample, those that occur in nature (e.g., an amino acid metabolite) orthose that are made synthetically (e.g., an α,α di-substituted aminoacid).

The term “polypeptide” encompasses two or more naturally ornon-naturally-occurring amino acids joined by a covalent bond (e.g., anamide bond). Polypeptides as described herein include full lengthproteins (e.g., fully processed proteins) as well as shorter amino acidssequences (e.g., fragments of naturally occurring proteins or syntheticpolypeptide fragments).

The term “halo” or “halogen” refers to fluorine, chlorine, bromine oriodine or a radical thereof.

The term “alkyl” refers to a hydrocarbon chain that is a straight chainor branched chain, containing the indicated number of carbon atoms. Forexample, C₁-C₁₀ indicates that the group has from 1 to 10 (inclusive)carbon atoms in it. In the absence of any numerical designation, “alkyl”is a chain (straight or branched) having 1 to 20 (inclusive) carbonatoms in it.

The term “alkylene” refers to a divalent alkyl (i.e., —R—).

The term “alkenyl” refers to a hydrocarbon chain that is a straightchain or branched chain having one or more carbon-carbon double bonds.The alkenyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive)carbon atoms in it. The term “lower alkenyl” refers to a C₂-C₆ alkenylchain. In the absence of any numerical designation, “alkenyl” is a chain(straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

The term “alkynyl” refers to a hydrocarbon chain that is a straightchain or branched chain having one or more carbon-carbon triple bonds.The alkynyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive)carbon atoms in it. The term “lower alkynyl” refers to a C₂-C₆ alkynylchain. In the absence of any numerical designation, “alkynyl” is a chain(straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring aresubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refersto alkyl substituted with an aryl. The term “arylalkoxy” refers to analkoxy substituted with aryl.

“Arylalkyl” refers to an aryl group, as defined above, wherein one ofthe aryl group's hydrogen atoms has been replaced with a C₁-C₅ alkylgroup, as defined above. Representative examples of an arylalkyl groupinclude, but are not limited to, 2-methylphenyl, 3-methylphenyl,4-methylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl2-propylphenyl, 3-propylphenyl, 4-propylphenyl, 2-butylphenyl,3-butylphenyl, 4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl,4-pentylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl,2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl, 2-sec-butylphenyl,3-sec-butylphenyl, 4-sec-butylphenyl, 2-t-butylphenyl, 3-t-butylphenyland 4-t-butylphenyl,

“Arylamido” refers to an aryl group, as defined above, wherein one ofthe aryl group's hydrogen atoms has been replaced with one or more—C(O)NH₂ groups. Representative examples of an arylamido group include2-C(O)NH2-phenyl, 3-C(O)NH₂-phenyl, 4-C(O)NH₂-phenyl, 2-C(O)NH₂-pyridyl,3-C(O)NH₂-pyridyl, and 4-C(O)NH₂-pyridyl,

“Alkylheterocycle” refers to a C₁-C₅ alkyl group, as defined above,wherein one of the C₁-C₅ alkyl group's hydrogen atoms has been replacedwith a heterocycle. Representative examples of an alkylheterocycle groupinclude, but are not limited to, —CH₂CH₂-morpholine, —CH₂CH₂-piperidine,—CH₂CH₂CH₂-morpholine, and —CH₂CH₂CH₂-imidazole.

“Alkylamido” refers to a C₁-C₅ alkyl group, as defined above, whereinone of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a—C(O)NH₂ group. Representative examples of an alkylamido group include,but are not limited to, —CH₂—C(O)NH₂, —CH₂CH₂—C(O)NH₂,—CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂CH₂C(O)NH₂,—CH₂CH(C(O)NH₂)CH₃, —CH₂CH(C(O)NH₂)CH₂CH₃, —CH(C(O)NH₂)CH₂CH₃ and—C(CH₃)₂CH₂C(O)NH₂.

“Alkanol” refers to a C₁-C₅ alkyl group, as defined above, wherein oneof the C₁-C₅ alkyl group's hydrogen atoms has been replaced with ahydroxyl group. Representative examples of an alkanol group include, butare not limited to, —CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂OH,—CH₂CH₂CH₂CH₂CH₂OH, —CH₂CH(OH)CH₃, —CH₂CH(OH)CH₂CH₃, —CH(OH)CH₃ and—C(CH₃)₂CH₂OH.

“Alkylcarboxy” refers to a C₁-C₅ alkyl group, as defined above, whereinone of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a—COOH group. Representative examples of an alkylcarboxy group include,but are not limited to, —CH₂COOH, —CH₂CH₂COOH, —CH₂CH₂CH₂COOH,—CH₂CH₂CH₂CH₂COOH, —CH₂CH(COOH)CH₃, —CH₂CH₂CH₂CH₂CH₂COOH,—CH₂CH(COOH)CH₂CH₃, —CH(COOH)CH₂CH₃ and —C(CH₃)₂CH₂COOH.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, whereinthe cycloalkyl group additionally is optionally substituted. Somecycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, andcyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring are substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to analkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refersto an alkoxy substituted with heteroaryl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring are substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to analkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refersto an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring are substituted by a substituent. Examples ofheterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl,morpholinyl, tetrahydrofuranyl, and the like.

The term “substituents” refers to a group “substituted” on an alkyl,cycloalkyl, aryl, heterocyclyl, or heteroaryl group at any atom of thatgroup. Suitable substituents include, without limitation, halo, hydroxy,mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy,thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy,alkanesulfonyl, alkylcarbonyl, and cyano groups.

In some embodiments, the compounds of this invention contain one or moreasymmetric centers and thus occur as racemates and racemic mixtures,single enantiomers, individual diastereomers and diastereomericmixtures. All such isomeric forms of these compounds are included in thepresent invention unless expressly provided otherwise. In someembodiments, the compounds of this invention are also represented inmultiple tautomeric forms, in such instances, the invention includes alltautomeric forms of the compounds described herein (e.g., if alkylationof a ring system results in alkylation at multiple sites, the inventionincludes all such reaction products). All such isomeric forms of suchcompounds are included in the present invention unless expresslyprovided otherwise. All crystal forms of the compounds described hereinare included in the present invention unless expressly providedotherwise.

As used herein, the terms “increase” and “decrease” mean, respectively,to cause a statistically significantly (i.e., p<0.1) increase ordecrease of at least 5%.

As used herein, the recitation of a numerical range for a variable isintended to convey that the invention may be practiced with the variableequal to any of the values within that range. Thus, for a variable whichis inherently discrete, the variable is equal to any integer valuewithin the numerical range, including the end-points of the range.Similarly, for a variable which is inherently continuous, the variableis equal to any real value within the numerical range, including theend-points of the range. As an example, and without limitation, avariable which is described as having values between 0 and 2 takes thevalues 0, 1 or 2 if the variable is inherently discrete, and takes thevalues 0.0, 0.1, 0.01, 0.001, or any other real values ≧0 and ≦2 if thevariable is inherently continuous.

As used herein, unless specifically indicated otherwise, the word “or”is used in the inclusive sense of “and/or” and not the exclusive senseof “either/or.”

The details of one or more particular embodiments of the invention areset forth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe description and drawings, and from the claims.

Peptidomimetic Macrocycles of the Invention

The present invention provides peptidomimetic macrocycles of Formula(I):

wherein:each A, C, D, and E is independently a natural or non-natural aminoacid;B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₄-CO—], [—NH-L₄-SO₂—], or [—NH-L₄-];R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo;R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, unsubstituted or substituted with R₅;L₁, L₂, L₃ and L₄ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene or [—R₄—K—R₄-]n, each being unsubstituted orsubstituted with R₅;K is O, S, SO, SO₂, CO, CO₂, or CONR₃;each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,unsubstituted or substituted with R₅, or part of a cyclic structure witha D residue;R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,unsubstituted or substituted with R₅, or part of a cyclic structure withan E residue;v is an integer from 1-1000;w is an integer from 1-1000;x is an integer from 0-10;y is an integer from 0-10;z is an integer from 0-10; andn is an integer from 1-5.

In some embodiments of the invention, x+y+z is at least 3. In otherembodiments of the invention, x+y+z is 3, 4, 5, 6, 7, 8, 9 or 10. Eachoccurrence of A, B, C, D or E in a macrocycle or macrocycle precursor ofthe invention is independently selected. For example, a sequencerepresented by the formula [A]_(x), when x is 3, encompasses embodimentswhere the amino acids are not identical, e.g. Gln-Asp-Ala as well asembodiments where the amino acids are identical, e.g. Gln-Gln-Gln. Thisapplies for any value of x, y, or z in the indicated ranges.

In some embodiments, the peptidomimetic macrocycle of the inventioncomprises a secondary structure which is an α-helix and R₈ is —H,allowing intrahelical hydrogen bonding.

In other embodiments, the length of the macrocycle-forming linker[-L₁-S-L₂-S L₃-] as measured from a first Cα to a second Cα is selectedto stabilize a desired secondary peptide structure, such as an α-helixformed by residues of the peptidomimetic macrocycle including, but notnecessarily limited to, those between the first Cα to a second Cα.

In some embodiments, the peptidomimetic macrocycle comprises at leastone α-helix motif. For example, A, B and/or C in the compound of FormulaI include one or more α-helices. As a general matter, α-helices includebetween 3 and 4 amino acid residues per turn. In some embodiments, theα-helix of the peptidomimetic macrocycle includes 1 to 5 turns and,therefore, 3 to 20 amino acid residues. In specific embodiments, theα-helix includes 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns. In someembodiments, the macrocycle-forming linker stabilizes an α-helix motifincluded within the peptidomimetic macrocycle. Thus, in someembodiments, the length of the macrocycle-forming linker [-L-S-L₂-S-L₃-]from a first Ca to a second Ca is selected to increase the stability ofan α-helix. In some embodiments, the macrocycle-forming linker spansfrom 1 turn to 5 turns of the α-helix. In some embodiments, themacrocycle-forming linker spans approximately 1 turn, 2 turns, 3 turns,4 turns, or 5 turns of the α-helix. In some embodiments, the length ofthe macrocycle-forming linker is approximately 5 Å to 9 Å per turn ofthe α-helix, or approximately 6 Å to 8 Å per turn of the α-helix. Wherethe macrocycle-forming linker spans approximately 1 turn of an α-helix,the length is equal to approximately 5 carbon-carbon bonds to 13carbon-carbon bonds, approximately 7 carbon-carbon bonds to 11carbon-carbon bonds, or approximately 9 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 2 turns of an α-helix, thelength is equal to approximately 8 carbon-carbon bonds to 16carbon-carbon bonds, approximately 10 carbon-carbon bonds to 14carbon-carbon bonds, or approximately 12 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 3 turns of an α-helix, thelength is equal to approximately 14 carbon-carbon bonds to 22carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20carbon-carbon bonds, or approximately 18 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 4 turns of an α-helix, thelength is equal to approximately 20 carbon-carbon bonds to 28carbon-carbon bonds, approximately 22 carbon-carbon bonds to 26carbon-carbon bonds, or approximately 24 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 5 turns of an α-helix, thelength is equal to approximately 26 carbon-carbon bonds to 34carbon-carbon bonds, approximately 28 carbon-carbon bonds to 32carbon-carbon bonds, or approximately 30 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 1 turn of an α-helix, thelinkage contains approximately 4 atoms to 12 atoms, approximately 6atoms to 10 atoms, or approximately 8 atoms. Where themacrocycle-forming linker spans approximately 2 turns of the α-helix,the linkage contains approximately 7 atoms to 15 atoms, approximately 9atoms to 13 atoms, or approximately 11 atoms. Where themacrocycle-forming linker spans approximately 3 turns of the α-helix,the linkage contains approximately 13 atoms to 21 atoms, approximately15 atoms to 19 atoms, or approximately 17 atoms. Where themacrocycle-forming linker spans approximately 4 turns of the α-helix,the linkage contains approximately 19 atoms to 27 atoms, approximately21 atoms to 25 atoms, or approximately 23 atoms. Where themacrocycle-forming linker spans approximately 5 turns of the α-helix,the linkage contains approximately 25 atoms to 33 atoms, approximately27 atoms to 31 atoms, or approximately 29 atoms. Where themacrocycle-forming linker spans approximately 1 turn of the α-helix, theresulting macrocycle forms a ring containing approximately 17 members to25 members, approximately 19 members to 23 members, or approximately 21members. Where the macrocycle-forming linker spans approximately 2 turnsof the α-helix, the resulting macrocycle forms a ring containingapproximately 29 members to 37 members, approximately 31 members to 35members, or approximately 33 members. Where the macrocycle-forminglinker spans approximately 3 turns of the α-helix, the resultingmacrocycle forms a ring containing approximately 44 members to 52members, approximately 46 members to 50 members, or approximately 48members. Where the macrocycle-fonnrming linker spans approximately 4turns of the α-helix, the resulting macrocycle forms a ring containingapproximately 59 members to 67 members, approximately 61 members to 65members, or approximately 63 members. Where the macrocycle-forminglinker spans approximately 5 turns of the α-helix, the resultingmacrocycle forms a ring containing approximately 74 members to 82members, approximately 76 members to 80 members, or approximately 78members.

In other embodiments, D and/or E are further modified in order tofacilitate cellular uptake. For example, lipidating or PEGylating apeptide facilitates in some embodiments cellular uptake, increasebioavailability, increase blood circulation, alter pharmacokinetics,decrease immunogenicity and/or decrease the needed frequency ofadministration.

The synthesis of the peptidomimetic macrocycles of the inventioninvolves a multi-step process that features the (1) synthesis of aprecursor peptide or peptidomimetic containing two free —SH moieties;and (2) then contacting the precursor with a bis-alkylating reagent togenerate two new covalent bonds.

Macrocycles or macrocycle precursors are synthesized, for example, bysolution phase or solid-phase methods, and can contain bothnaturally-occurring and non-naturally-occurring amino acids. See, forexample, Hunt, “The Non-Protein Amino Acids” in Chemistry andBiochemistry of the Amino Acids, edited by G. C. Barrett, Chapman andHall, 1985. In some embodiments, the thiol moieties are the side chainsof the amino acid residues L-cysteine, D-cysteine, α-methyl-L cysteine,α-methyl-D-cysteine. L-homocysteine, D-homocysteine,α-methyl-L-homocysteine or α-methyl-D-homocysteine. The bis-alkylatingreagent is of the general formula X-L₂-Y wherein L₂ is a linker moietyand X and Y are leaving groups that are displaced by —SH moieties toform bonds with L₂. In some embodiments, X and Y are halogens such as I,Br, or Cl.

In one embodiment, the peptidomimetic macrocycle exhibits improvedbiological properties such as increased structural stability, increasedaffinity for a target increased resistance to proteolytic degradationand/or increased cell penetrance when compared to the precursor peptideor peptidomimetic. In another embodiment, the peptidomimetic macrocyclecomprises one or more α-helices in aqueous solutions and/or exhibits anincreased degree of α-helicity when compared to the precursor peptide orpeptidomimetic. The method provides a route towards the synthesis of alibrary of peptidomimetic macrocycles by varying the X-L₂-Y reagent, andin some embodiments the linker element L₂ is optimized to improve thebiological or pharmacological properties of the resultant peptidomimeticmacrocycle.

In some embodiments, the macrocycle-forming linker increases cellpermeability of the peptidomimetic macrocycle. Thus, in someembodiments, the macrocycle-forming linker increases the overallhydrophobicity of the peptidomimetic macrocycle relative to theprecursor peptide or peptidomimetic.

Any protein or polypeptide with a known primary amino acid sequencewhich contains a secondary structure believed to impart biologicalactivity is the subject of the present invention. For example, thesequence of the polypeptide can be analyzed and the sulfhydrylcontaining amino acid analogs of the invention can be substituted at theappropriate positions. The appropriate positions are determined byascertaining which molecular surface(s) of the secondary structure is(are) required for biological activity and, therefore, across whichother surface(s) the macrocycle forming linkers of the invention canform a macrocycle without sterically blocking the surface(s) requiredfor biological activity. Such determinations are made using methods suchas X-ray crystallography of complexes between the secondary structureand a natural binding partner to visualize residues (and surfaces)critical for activity; by sequential mutagenesis of residues in thesecondary structure to functionally identify residues (and surfaces)critical for activity; or by other methods. By such determinations, theappropriate amino acids are substituted with the amino acids analogs andmacrocycle-forming linkers of the invention. For example, for anα-helical secondary structure, one surface of the helix (e.g., amolecular surface extending longitudinally along the axis of the helixand radially 45-135 about the axis of the helix) may be required to makecontact with another biomolecule in vivo or in vitro for biologicalactivity. In such a case, a macrocycle-forming linker is designed tolink two α-carbons of the helix while extending longitudinally along thesurface of the helix in the portion of that surface not directlyrequired for activity.

In some embodiments of the invention, the peptide sequence is derivedfrom the BCL-2 family of proteins. The BCL-2 family is defined by thepresence of up to four conserved BCL-2 homology (BH) domains designatedBH1, BH2, BH3, and BH4, all of which include α-helical segments(Chittenden el al. (1995), EMBO 14:5589; Wang et al. (1996), Genes Dev.10:2859). Anti-apoptotic proteins, such as BCL-2 and BCL-X_(L), displaysequence conservation in all BH domains. Pro-apoptotic proteins aredivided into “multidomain” family members (e.g., BAK, BAX), whichpossess homology in the BH1, BH2, and BH3 domains, and “BH3-domain only”family members (e.g., BID. BAD, BIM, BIK, NOXA, PUMA), that containsequence homology exclusively in the BH3 amphipathic α-helical segment.BCL-2 family members have the capacity to form homo- and heterodimers,suggesting that competitive binding and the ratio between pro- andanti-apoptotic protein levels dictates susceptibility to death stimuli.Anti-apoptotic proteins function to protect cells from pro-apoptoticexcess, i.e., excessive programmed cell death. Additional “security”measures include regulating transcription of pro-apoptotic proteins andmaintaining them as inactive conformers, requiring either proteolyticactivation, dephosphorylation, or ligand-induced conformational changeto activate pro-death functions. In certain cell types, death signalsreceived at the plasma membrane trigger apoptosis via a mitochondrialpathway. The mitochondria can serve as a gatekeeper of cell death bysequestering cytochrome c, a critical component of a cytosolic complexwhich activates caspase 9, leading to fatal downstream proteolyticevents. Multidomain proteins such as BCL-2/BCL-X_(L) and BAK/BAX playdueling roles of guardian and executioner at the mitochondrial membrane,with their activities further regulated by upstream BH3-only members ofthe BCL-2 family. For example, BID is a member of the BH3-domain onlyfamily of pro-apoptotic proteins, and transmits death signals receivedat the plasma membrane to effector pro-apoptotic proteins at themitochondrial membrane. BID has the capability of interacting with bothpro- and anti-apoptotic proteins, and upon activation by caspase 8,triggers cytochrome c release and mitochondrial apoptosis. Deletion andmutagenesis studies determined that the amphipathic α-helical BH3segment of pro-apoptotic family members functions as a death domain andthus represents a critical structural motif for interacting withmultidomain apoptotic proteins. Structural studies have demonstratedthat the BH3 helix interacts with anti-apoptotic proteins by insertinginto a hydrophobic groove formed by the interface of BH1, 2 and 3domains. Activated BID can be bound and sequestered by anti-apoptoticproteins (e.g., BCL-2 and BCL-X_(L)) and can trigger activation of thepro-apoptotic proteins BAX and BAK, leading to cytochrome c release anda mitochondrial apoptosis program. BAD is also a BH3-domain onlypro-apoptotic family member whose expression triggers the activation ofBAX/BAK. In contrast to BID, however, BAD displays preferential bindingto anti-apoptotic family members, BCL-2 and BCL-X_(L). Whereas the BADBH3 domain exhibits high affinity binding to BCL-2, BAD BH3 peptide isunable to activate cytochrome c release from mitochondria in vitro,suggesting that BAD is not a direct activator of BAX/BAK. Mitochondriathat over-express BCL-2 are resistant to BID-induced cytochrome crelease, but co-treatment with BAD can restore BID sensitivity.Induction of mitochondrial apoptosis by BAD appears to result fromeither: (1) displacement of BAX/BAK activators, such as BID and BID-likeproteins, from the BCL-2/BCL-XL binding pocket, or (2) selectiveoccupation of the BCL-2/BCL-XL binding pocket by BAD to preventsequestration of BID-like proteins by anti-apoptotic proteins. Thus, twoclasses of BH3-domain only proteins have emerged. BID-like proteins thatdirectly activate mitochondrial apoptosis, and BAD-like proteins, thathave the capacity to sensitize mitochondria to BID-like pro-apoptoticsby occupying the binding pockets of multidomain anti-apoptotic proteins.Various α-helical domains of BCL-2 family member proteins amendable tothe methodology disclosed herein have been disclosed (Walensky et al.(2004), Science 305:1466; and Walensky et al., U.S. Patent PublicationNo. 2005/0250680, the entire disclosures of which are incorporatedherein by reference).

In other embodiments, the peptide sequence is derived from the tumorsuppressor p53 protein which binds to the oncogene protein MDM2. TheMDM2 binding site is localized within a region of the p53 tumorsuppressor that forms an α helix. In U.S. Pat. No. 7,083,983, the entirecontents of which are incorporated herein by reference, Lane et al.disclose that the region of p53 responsible for binding to MDM2 isrepresented approximately by amino acids 13-31 (PLSQETFSDLWKLLPENNV) ofmature human P53 protein. Other modified sequences disclosed by Lane arealso contemplated in the instant invention. Furthermore, the interactionof p53 and MDM2 has been discussed by Shair et al. (1997), Chem. & Biol.4:791, the entire contents of which are incorporated herein byreference, and mutations in the p53 gene have been identified invirtually half of all reported cancer cases. As stresses are imposed ona cell, p53 is believed to orchestrate a response that leads to eithercell-cycle arrest and DNA repair, or programmed cell death. As well asmutations in the p53 gene that alter the function of the p53 proteindirectly, p53 can be altered by changes in MDM2. The MDM2 protein hasbeen shown to bind to p53 and disrupt transcriptional activation byassociating with the transactivation domain of p53. For example, an 11amino-acid peptide derived from the transactivation domain of p53 formsan amphipathic α-helix of 2.5 turns that inserts into the MDM2 crevice.Thus, in some embodiments, novel α-helix structures generated by themethod of the present invention are engineered to generate structuresthat bind tightly to the helix acceptor and disrupt nativeprotein-protein interactions. These structures are then screened usinghigh throughput techniques to identify optimal small molecule peptides.The novel structures that disrupt the MDM2 interaction are useful formany applications, including, but not limited to, control of soft tissuesarcomas (which over-expresses MDM2 in the presence of wild type p53).These cancers are then, in some embodiments, held in check with smallmolecules that intercept MDM2, thereby preventing suppression of p53.Additionally, in some embodiments, small molecules disrupters ofMDM2-p53 interactions are used as adjuvant therapy to help control andmodulate the extent of the p53 dependent apoptosis response inconventional chemotherapy.

A non-limiting exemplary list of suitable peptides for use in thepresent invention is given below:

TABLE 1¹ Name Sequence (bold = critical residues) Cross-linked Sequence(X = x-link residue) BH3 peptides BID-BH3 QEDIIRNIARHLAQVGDSMDRSIPPQEDIIRNIARHLA X VGDX MDRSIPP BIM-BH3 DNRPEIWIAQELRRIGDEFNAYYARDNRPEIWIAQELR X IGDX FNAYYAR BAD-BH3 NLWAAQRYGRELRRMSDEFVDSFKKNLWAAQRYGRELR X MSD X FVDSFKK PUMA-BH3 EEQWAREIGAQLRRMADDLNAQYEREEQWAREIGAQLR X MA D XLNAQYER Hrk-BH3 RSSAAQLTAARLKALGDELHQRTMRSSAAQLTAARLK X LGDX LHQRTM NOXAA-BH3 AELPPEFAAQLRKIGDKVYCTWAELPPEFAAQLR X IGDX VYCTW NOXAB-BH3 VPADLKBECAQLRRIGDKVNLRQKLVPADLKDECAQLR X IGDX VNLRQKL BMF-BH3 QHRAEVQIARKLQCIADQFHRLHTQHRAEVQIARKLQ X IADX FHRLHT BLK-BH3 SSAAQLTAARLKALGDELHQRT SSAAQLTAARLKX LGDX LHQRT BIK-BH3 CMEGSDALALRLACIGDEMDVSLRA CMEGSDALALRLA X IGDXMDVSLRA Bnip3 DIERRKEVESILKKNSDWIWDWSS DIERRKEVESILK X NSDX IWDWSSBOK-BH3 GRLAEVCAVLLRLGDELEMIRP GRLAEVCAVLL X LGDX LEMIRP BAX-BH3PQDASTKKSECLKRIGDELDSNMEL PQDASTKKSECLK X IGDX LDSNMEL BAK-BH3PSSTMGQVGRQLAIIGDDINRR PSSTMGQVGRQLA X IGDX INRR BCL2L1-BH3KQALREAGDEFELR KQALR X AGDX FELR BCL2-BH3 LSPPYVHLALALRQAGDDFSRRLSPPVVHLALALR X AGDX FSRR BCL-XL-BH3 EVIPMAAVKQALREAGDEFELRYEVIPMAAVKQALR X AGDX FELRY BCL-W-BH3 PADPLHQAMRAAGDEFETRF PADPLHQAMR XAGDX FETRF MCL1-BH3 ATSRKLETLRRVGDGVQRNHETA ATSRKLETLR X VGDX VQRNHETAMTD-BH3 LAEVCTVLLRLGDELEQIR LAEVCTVLL X LGDX LEQIR MAP-1-BH3MTVGELSRALGHENGSLDP MTVGELSRALG X ENG X LDP NIX-BH3VVEGEKEVEALKKSADWVSDWS VVEGEKEVEALK X SAD X VSDWS 4ICD(ERBB4)-BH3SMARDPQRYLVIQGDDRMKL SMARDPQRYLVX Q GDX RMKL ¹Peptide sequences listedin Table 1 are human sequences which target the BH3 binding site and areimplicated in cancers, autoimmune disorders, metabolic diseases andother human disease conditions.

TABLE 2¹ Name Sequence (bold = critical residues) Cross-linked Sequence(X = x-link residue) BH3 peptides BID-BH3 QEDIIRNIARHLAQVGDSMDRSIPPQEDIIRNI X RHLX QVGDSMDRSIPP BIM-BH3 DNRPEIWIAQELRRIGDEFNAYYAR DNRPEIWIX QELX RIGDEFNAYYAR BAD-BH3 NLWAAQRYGRELRRMSDEFVDSFKK NLWAAQRY X RELXRMSDEFVDSFKK PUMA-BH3 EEQWAREIGAQLRRMADDLNAQYER EEQWAREI X AQLXRMADDLNAQYER Hrk-BH3 RSSAAQLTAARLKALGDELHQRTM RSSAAQLT X ARLXALGDELHQRTM NOXAA-BH3 AELPPEFAAQLRKIGDKVYCTW AELPPEF X AQLX KIGDKVYCTWNOXAB-BH3 VPADLKDECAQLRRIGDKVNLRQKL VPADLKDE X AQLX RIGDKVNLRQKL BMF-BH3QHRAEVQIARKLQCIADQFHRLHT QHRAEVQI X RKLX CIADQFHRLHT BLK-BH3SSAAQLTAARLKALGDELHQRT SSAAQLT X ARLX ALGDELHQRT BIK-BH3CMEGSDALALRLACIGDEMDVSLRA CMEGSDAL X LRLX CIGDEMDVSLRA Bnip3DIERRKEVESILKKNSDWIWDWSS DIERRKEV X SILX KNSDWIWDWSS BOK-BH3GRLAEVCAVLLRLGDELEMIRP GRLAEV X AVLX RLGDELEMIRP BAX-BH3PQDASTKKSECLKRIGDELDSNMEL PQDASTKK X ECLX RIGDELDSNMEL BAK-BH3PSSTMGQVGRQLAIIGDDINRR PSSTMGQV X RQLX IIGDDINRR BCL2L1-BH3KQALREAGDEFELR X QALX EAGDEFELR BCL2-BH3 LSPPVVHLALALRQAGDDFSRR LSPPVVHLX LALX QAGDDFSRR BCL-XL-BH3 EVIPMAAVKQALREAGDEFELRY EVIPMAAV X QALXEAGDEFELRY BCL-W-BH3 PADPLHQAMRAAGDEFETRF PADPL X QAM X AAGDEFETRFMCL1-BH3 ATSRKLETLRRVGDGVQRNHETA ATSRK X ETLX RVGDGVQRNHETA MTD-BH3LAEVCTVLLRLGDELEQIR LAEV X TVLX RLGDELEQIR MAP-1-BH3 MTVGELSRALGHENGSLDPMTVGEL X RALX HENGSLDP NIX-BH3 VVEGEKEVEALKKSADWVSDWS VVEGEKE X EALXKSADWVSDWS 4ICD(ERBB4)-BH3 SMARDPQRYLVIQGDDRMKL SMARDP X RYLX IQGDDRMKL¹Peptide sequences listed in Table 2 are human sequences which targetthe BH3 binding site and are implicated in cancers, autoimmunedisorders, metabolic diseases and other human disease conditions.

TABLE 3¹ Name Sequence (bold = critical residues) Cross-linked Sequence(X = x-link residue) P53 peptides hp53 peptide_veryshortLSQETFSDLWKLLPEN X SQE X FSDLWKLLPEN hp53 peptide_shortPPLSQETFSDLWKLLPENN PP X SQE X FSDLWKLLPENN hp53-P27S peptide-shortPPLSQETFSDLWKLLSENN PP X SQE X FSDLWKLLSENN hp53 peptide_LongDPSVEPPLSQETFSDLWKLLPENNVLSPLP DPSVEPP X SQE X FSDLWKLLPENNVLSPLPhp53-P27S peptide_Long DPSVEPPLSQETFSDLWKLLSENNVLSPLP DPSVEPP X SQE XFSDLWKLLSENNVLSPLP hp53 peptide_veryshort LSQETFSDLWKLLPEN LSQETFSDLWXLLP X N hp53 peptide_short PPLSQETFSDLWKLLPENN PPLSQETFSDLWX LLP X NNhp53-P27S peptide-short PPLSQETFSDLWKLLSENN PPLSQETFSDLWX LLS X NN hp53peptide_Long DPSVEPPLSQETFSDLWKLLPENNVLSPLP DPSVEPPLSQETFSDLWX LLP XNNVLSPLP hp53-P27S peptide_Long DPSVEPPLSQETFSDLWKLLSENNVLSPLPDPSVEPPLSQETFSDLWX LLS X NNVLSPLP ¹Peptide sequences listed in Table 3are human sequences which target the p53 binding site of mdm2/x and areimplicated in cancers.

TABLE 4¹ Cross-linked Sequence Sequence (bold = critical (X = x-linkName residues) residue) GPCR peptide ligands Angiotensin II DRVYIHPF DRXYXHPF Bombesin EQRLGNQWAVGHLM EQRLGN XWAVGHLX Bradykinin RPPGFSPFR RPPX FSPFRX C5a ISHKDMQLGR ISHKDMXLGRX C3a ARASHLGLAR ARASHLXLARXα-melanocyte SYSMEHFRWGKPV SYSM XHFRWX KPV stimulating hormone ¹Peptidesequences listed in Table 4 are sequences which target human Gprotein-coupled receptors and are implicated in numerous human diseaseconditions (Tyndall, J. D. A. et al. Chem. Rev. 2005, 105, 793-826).

Methods of Synthesizing the Peptidomimetic Macrocycles of the Invention

Methods of synthesizing the peptidomimetic macrocycles of the inventionare disclosed herein. Alternative but equivalent protecting groups,leaving groups or reagents are substituted, and certain of the syntheticsteps are performed in alternative sequences or orders to produce thedesired compounds. Synthetic chemistry transformations and protectinggroup methodologies (protection and deprotection) useful in synthesizingthe compounds described herein include, for example, those such asdescribed in Larock, Comprehensive Organic Transformations, VCHPublishers (1989); Greene and Wuts, Protective Groups in OrganicSynthesis, 2d. Ed., John Wiley and Sons (1991); Fieser and Fieser,Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons(1994); and Paquette, ed., Encyclopedia of Reagents for OrganicSynthesis, John Wiley and Sons (1995), and subsequent editions thereof.

The peptidomimetic macrocycles of the invention are made, for example,by chemical synthesis methods, such as described in Fields et al.,Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H.Freeman & Co., New York. N.Y., 1992, p. 77. Hence, for example, peptidesare synthesized using the automated Merrifield techniques of solid phasesynthesis with the amine protected by either tBoc or Fmoc chemistryusing side chain protected amino acids on, for example, an automatedpeptide synthesizer (e.g., Applied Biosystems (Foster City, Calif.),Model 430A, 431, or 433).

One manner of producing the precursor peptides and peptidomimeticsdescribed herein uses solid phase peptide synthesis (SPPS). TheC-terminal amino acid is attached to a cross-linked polystyrene resinvia an acid labile bond with a linker molecule. This resin is insolublein the solvents used for synthesis, making it relatively simple and fastto wash away excess reagents and by-products. The N-terminus isprotected with the Fmoc group, which is stable in acid, but removable bybase. Side chain functional groups are protected as necessary with basestable, acid labile groups.

Longer precursor peptides are produced, for example, by conjoiningindividual synthetic peptides using native chemical ligation.Alternatively, the longer synthetic peptides are biosynthesized by wellknown recombinant DNA and protein expression techniques. Such techniquesare provided in well-known standard manuals with detailed protocols. Toconstruct a gene encoding a precursor peptide of this invention, theamino acid sequence is reverse translated to obtain a nucleic acidsequence encoding the amino acid sequence, preferably with codons thatare optimum for the organism in which the gene is to be expressed. Next,a synthetic gene is made, typically by synthesizing oligonucleotideswhich encode the peptide and any regulatory elements, if necessary. Thesynthetic gene is inserted in a suitable cloning vector and transfectedinto a host cell. The peptide is then expressed under suitableconditions appropriate for the selected expression system and host. Thepeptide is purified and characterized by standard methods.

The precursor peptides and peptidomimetics are made, for example, in ahigh-throughput, combinatorial fashion using, for example, ahigh-throughput polychannel combinatorial synthesizer (e.g., Model Apex396 multichannel peptide synthesizer from AAPPTEC, Inc., Louisville,Ky.).

The discussion below is offered to illustrate certain of the diversemethods available for use in assembling the compounds of the invention.However, the discussion is not intended to limit the scope of reactionsor reaction sequences that are useful in preparing the compounds of thepresent invention.

The following synthetic schemes are provided solely to illustrate thepresent invention and are not intended to limit the scope of theinvention, as described herein. To simplify the drawings, theillustrative schemes depict amino acid analogs derived from L- orD-cysteine, in which L₁ and L₃ are both —(CH₂)—. However, as notedthroughout the detailed description above, many other amino acid analogscan be employed in which L₁ and L₃ can be independently selected fromthe various structures disclosed herein. The symbols “[AA]_(m)”,“[AA]_(n)”. “[AA]_(o)” represent a sequence of amide bond-linkedmoieties such as natural or unnatural amino acids. As describedpreviously, each occurrence of “AA” is independent of any otheroccurrence of “AA”, and a formula such as “[AA],” encompasses, forexample, sequences of non-identical amino acids as well as sequences ofidentical amino acids.

In this first general method, the peptidomimetic precursor contains two—SH moieties and is synthesized by solid-phase peptide synthesis (SPPS)using commercially available N-α-Fmoc amino acids such asN-α-Fmoc-S-trityl-L-cysteine or N-α-Fmoc-S-trityl-D-cysteine.Alpha-methylated versions of D-cysteine or L-cysteine are generated byknown methods (Seebach et al. (1996), Angew. Chem. Int. Ed. Engl.35:2708-2748, and references therein) and then converted to theappropriately protected N-α-Fmoc-S-trityl monomers by known methods(“Bioorganic Chemistry: Peptides and Proteins”, Oxford University Press,New York: 1998, the entire contents of which are incorporated herein byreference). The precursor peptidomimetic is then deprotected and cleavedfrom the solid-phase resin by standard conditions (e.g., strong acidsuch as 95% TFA). The precursor peptidomimetic is reacted as a crudemixture or is purified prior to reaction with X-L₂-Y in organic oraqueous solutions. In some embodiments the alkylation reaction isperformed under dilute conditions (i.e. 0.15 mmol/L) to favormacrocyclization and to avoid polymerization. In some embodiments, thealkylation reaction is performed in organic solutions such as liquid NH₃(Mosberg et al. (1985), J. Am. Chem. Soc. 107:2986-2987; Szewczuk et al.(1992), Int. J. Peptide Protein Res. 40:233-242), NH₃/MeOH, or NH₃/DMF(Or et al. (1991), J. Org. Chem. 56:3146-3149). In other embodiments,the alkylation is performed in an aqueous solution such as 6Mguanidinium HCL, pH 8 (Brunel et al. (2005), Chem. Commun.(20):2552-2554). In other embodiments, the solvent used for thealkylation reaction is DMF or dichloroethane.

In this second general method, the precursor peptidomimetic contains twoor more —SH moieties, of which two are specially protected to allowtheir selective deprotection and subsequent alkylation for macrocycleformation. The precursor peptidomimetic is synthesized by solid-phasepeptide synthesis (SPPS) using commercially available N-α-Fmoc aminoacids such as N-α-Fmoc-S-p-methoxytrityl-L-cysteine orN-α-Fmoc-S-p-methoxytrityl-D-cysteine. Alpha-methylated versions ofD-cysteine or L-cysteine are generated by known methods (Seebach et al.(1996). Angew. Chem. Int. Ed. Engl. 35:2708-2748, and referencestherein) and then converted to the appropriately protectedN-α-Fmoc-S-p-methoxytrityl monomers by known methods (BioorganicChemistry: Peptides and Proteins, Oxford University Press, New York:1998, the entire contents of which are incorporated herein byreference). The Mmt protecting groups of the peptidomimetic precursorare then selectively cleaved by standard conditions (e.g., mild acidsuch as 1% TFA in DCM). The precursor peptidomimetic is then reacted onthe resin with X-L₂-Y in an organic solution. For example, the reactiontakes place in the presence of a hindered base such asdiisopropylethylamine. In some embodiments, the alkylation reaction isperformed in organic solutions such as liquid NH₃ (Mosberg et al.(1985), J. Am. Chem. Soc. 107:2986-2987; Szewczuk et al. (1992), Int. JPeptide Protein Res. 40:233-242), NH₃/MeOH or NH₃/DMF (Or et al. (1991),J. Org. Chem. 56:3146-3149). In other embodiments, the alkylationreaction is performed in DMF or dichloroethane. The peptidomimeticmacrocycle is then deprotected and cleaved from the solid-phase resin bystandard conditions (e.g., strong acid such as 95% TFA).

In this third general method, the peptidomimetic precursor contains twoor more —SH moieties, of which two are specially protected to allowtheir selective deprotection and subsequent alkylation for macrocycleformation. The peptidomimetic precursor is synthesized by solid-phasepeptide synthesis (SPPS) using commercially available N-ca-Fmoc aminoacids such as N-α-Fmoc-S-p-methoxytrityl-L-cysteine,N-α-Fmoc-S-p-methoxytrityl-D-cysteine, N-α-Fmoc-S-S-t-butyl-L-cysteine,and N-α-Fmoc-S-S-t-butyl-D-cysteine. Alpha-methylated versions ofD-cysteine or L-cysteine are generated by known methods (Seebach et al.(1996), Angew. Chem. Int. Ed. Engl. 35:2708-2748, and referencestherein) and then converted to the appropriately protectedN-α-Fmoc-S-p-methoxytrityl or N-α-Fmoc-S-S-t-butyl monomers by knownmethods (Bioorganic Chemistry: Peptides and Proteins, Oxford UniversityPress, New York: 1998, the entire contents of which are incorporatedherein by reference). The S-S-tButyl protecting group of thepeptidomimetic precursor is selectively cleaved by known conditions(e.g., 20% 2-mercaptoethanol in DMF, reference: Galande et al. (2005),J. Comb. Chem. 7:174-177). The precursor peptidomimetic is then reactedon the resin with a molar excess of X-L₂-Y in an organic solution. Forexample, the reaction takes place in the presence of a hindered basesuch as diisopropylethylamine. The Mint protecting group of thepeptidomimetic precursor is then selectively cleaved by standardconditions (e.g., mild acid such as 1% TFA in DCM). The peptidomimeticprecursor is then cyclized on the resin by treatment with a hinderedbase in organic solutions. In some embodiments, the alkylation reactionis performed in organic solutions such as NH₃/MeOH or NH₃/DMF (Or et al.(1991), J. Org. Chem. 56:3146-3149). The peptidomimetic macrocycle isthen deprotected and cleaved from the solid-phase resin by standardconditions (e.g., strong acid such as 95% TFA).

In this fourth general method, the peptidomimetic precursor contains twoL-cysteine moieties. The peptidomimetic precursor is synthesized byknown biological expression systems in living cells or by known invitro, cell-free, expression methods. The precursor peptidomimetic isreacted as a crude mixture or is purified prior to reaction with X-L2-Yin organic or aqueous solutions. In some embodiments the alkylationreaction is performed under dilute conditions (i.e. 0.15 mmol/L) tofavor macrocyclization and to avoid polymerization. In some embodiments,the alkylation reaction is performed in organic solutions such as liquidNH₃ (Mosberg et al. (1985), J. Am. Chem. Soc. 107:2986-2987; Szewczuk etal. (1992), Int. J. Peptide Protein Res. 40:233-242), NH₃/MeOH, orNH₃/DMF (Or et al. (1991), J. Org. Chem. 56:3146-3149). In otherembodiments, the alkylation is performed in an aqueous solution such as6M guanidinium HCL, pH 8 (Brunel et al. (2005), Chem. Commun.(20):2552-2554). In other embodiments, the alkylation is performed inDMF or dichloroethane. In another embodiment, the alkylation isperformed in non-denaturing aqueous solutions, and in yet anotherembodiment the alkylation is performed under conditions that favorα-helical structure formation. In yet another embodiment, the alkylationis performed under conditions that favor the binding of the precursorpeptidomimetic to another protein, so as to induce the formation of thebound α-helical conformation during the alkylation.

Various embodiments for X and Y are envisioned which are suitable forreacting with thiol groups. In general, each X or Y is independently beselected from the general category shown in Table 5. For example, X andY are halides such as —Cl, —Br or —I.

TABLE 5 Examples of Reactive Groups Capable of Reacting with ThiolGroups and Resulting Linkages X or Y Resulting Covalent Linkageacrylamide Thioether halide (e.g. alkyl or aryl halide) Thioethersulfonate Thioether aziridine Thioether epoxide Thioether haloacetamideThioether maleimide Thioether sulfonate ester Thioether

Table 6 shows exemplary macrocycles of the invention. For the examplesshown in this table, a corresponding non-macrocyclic polypeptide is theBID BH3 polypeptide sequence fragment DIIRNIARHLAQVGDSMDRSI. “N_(L)”represents norleucine and replaces a methionine residue. It isenvisioned that similar linkers are used to synthesize peptidomimeticmacrocycles based on the polypeptide sequences disclosed in Table 1through Table 4.

TABLE 6 Examples of Peptidomimetic Macrocycles of the Invention

MW = 2449

MW = 2435

MW = 2497

MW = 2503

MW = 2447

MW = 2447

MW = 2477

MW = 2463

MW = 2525

MW = 2531

MW = 2475

MW = 2475 For the examples shown in this table, a correspondingnon-macrocyclic polypeptide is the BID BH3 polypeptide sequence fragmentDIIRNIARHLAQVGDSMDRSI. “N_(L)” represents norleucine.

Amino Acid Analogs

The present invention contemplates the use of both naturally-occurringand non-naturally-occurring amino acids and amino acid analogs in thesynthesis of the peptidomimetic macrocycles described above. Any aminoacid or amino acid analog amenable to the synthetic methods employed forthe synthesis of stable bis-sulfhydryl containing peptidomimeticmacrocycles can be used in the present invention. For example, cysteineis contemplated as a useful amino acid in the present invention.However, sulfur containing amino acids other than cysteine that containa different amino acid side chain are also useful in the invention. Forexample, cysteine contains one methylene unit between the α-carbon ofthe amino acid and the terminal —SH of the amino acid side chain. Theinvention also contemplates the use of amino acids with multiplemethylene units between the α-carbon and the terminal —SH. Non-limitingexamples include L-homocysteine. D-homocysteine. α-methyl-L-homocysteineand α-methyl-D-homocysteine. In some embodiments the amino acids andamino acid analogs are of the D-configuration. In other embodiments theyare of the L-configuration. In some embodiments, some of the amino acidsand amino acid analogs contained in the peptidomimetic are of theD-configuration while some of the amino acids and amino acid analogs areof the L-configuration. In some embodiments the amino acid analogs areα,α-disubstituted, such as α-methyl-L-cysteine and α-methyl-D-cysteine.In some embodiments the amino acid analogs are N-alkylated, e.g.,N-methyl-L-cysteine and N-methyl-D-cysteine.

Other amino acid analogs useful in the present invention for formingpeptidomimetic macrocycles are compounds of Formula IIa:

wherein:R₁ is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,heteroalkyl, or heterocycloalkyl;L₁ is independently alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene or[—R₄—K—R₄-]n, unsubstituted or substituted with R₅;K is O, S, SO, SO₂, CO, CO₂, or CONR₃;R₄ is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,heterocycloalkylene, arylene, or heteroarylene;R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆,—CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent;R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heteroalkylalkyl, heterocyclyalkyl, a fluorescentmoiety, a radioisotope or a therapeutic agent;R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl,heteroalkylalkyl, or heterocyclyalkyl;R₉ and R₁₀ are independently —H or a protecting group suitable forpeptide synthesis;n is an integer from 1 to 5;

Q is S; and

P is —H, -trityl, p-methoxytrityl, —S t-butyl, or any other protectinggroup suitable for peptide synthesis; or Q and P when taken togetherform a moiety capable of undergoing chemical transformation into an —SHgroup.

In some embodiments either the —NH or the —SH moieties of the amino acidare protected. In other embodiments both moieties are protected, forexample protecting groups for either the —NH and the —SH moieties inamino acids. Non-limiting examples of such —NH protecting groups are-Fmoc and -Boc. Non-limiting examples of —SH protecting groups are-trityl, p-methoxytrityl, and —S t-butyl. In other embodiments, theamino acid is not protected prior to synthesis of the peptidomimeticmacrocycle.

TABLE 7 Exemplary amino acids of the invention.

Macrocycle-Forming Linkers

The present invention includes macrocycle-forming linkers used to linktwo or more —SH moieties in the peptidomimetic precursors to form thepeptidomimetic macrocycles of the invention. As described above, themacrocycle-forming linkers impart conformational rigidity, increasedmetabolic stability and/or increased cell penetrability. Furthermore, insome embodiments, the macrocycle-forming linkages stabilize theα-helical secondary structure of the peptidomimetic macrocycles. Themacrocycle-forming linkers are of the formula X-L₂-Y, wherein both X andY are the same or different moieties, as defined above. Both X and Yhave the chemical characteristics that allow one macrocycle-forminglinker -L₂- to bis alkylate the bis-sulfhydryl containing peptidomimeticprecursor. As defined above, the linker -L₂- includes alkylene,alkenylene, alkynylene, heteroalkylene, cycloalkylene,heterocycloalkylene, cycloarylene, or heterocycloarylene, or —R₄—K—R₄—,all of which can be optionally substituted with an R₅ group, as definedabove. Furthermore, one to three carbon atoms within themacrocycle-forming linkers -L₂-, other than the carbons attached to the—SH of the sulfhydryl containing amino acid, are optionally substitutedwith a heteroatom such as N, S or O.

The L₂ component of the macrocycle-forming linker X-L₂-Y may be variedin length depending on, among other things, the distance between thepositions of the two amino acid analogs used to form the peptidomimeticmacrocycle. Furthermore, as the lengths of L₁ and/or L₃ components ofthe macrocycle-forming linker are varied, the length of L₂ can also bevaried in order to create a linker of appropriate overall length forforming a stable peptidomimetic macrocycle. For example, if the aminoacid analogs used are varied by adding an additional methylene unit toeach of L₁ and L₃, the length of L₂ are decreased in length by theequivalent of approximately two methylene units to compensate for theincreased lengths of L₁ and L₃.

In some embodiments, L₂ is an alkylene group of the formula —(CH₂)_(n)—,where n is an integer between about 1 and about 15. For example, n is 1,2, 3, 4, 5, 6, 7, 8, 9 or 10. In other embodiments, L₂ is an alkenylenegroup. In still other embodiments, L₂ is an aryl group.

Table X shows additional embodiments of X-L₂-Y groups.

TABLE 8 Exemplary X—L₂—Y groups of the invention.

Each X and Y in this Table, are, for example, independently Cl—, Br— orI—.

Kits

In another aspect, the present invention further provides kitscomprising amino acid analogs and/or macrocycle-forming linkers asdescribed herein.

In one embodiment the kit contains a) a compound of Formulas IIa and acompound of Formula IIb:

-   -   wherein        R₁ is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,        heteroalkyl, or heterocycloalkyl, unsubstituted or substituted        with halo-;        R₂ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,        heteroalkyl, or heterocycloalkyl, unsubstituted or substituted        with halo-;        L₁ and L₃ are independently alkylene, alkenylene, alkynylene,        heteroalkylene, cycloalkylene, heterocycloalkylene,        cycloarylene, or heterocycloarylene or [—R₄—K—R₄-]n, each being        unsubstituted or substituted with R₅;        K is O, S, SO, SO₂, CO, CO₂, or CONR₃;        R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,        cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;        each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆,        —SOR₆, —SO₂—CO₂R₆, —R₆, a fluorescent moiety, a radioisotope, or        a therapeutic agent;        each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,        cycloalkylalkyl, heteroalkylalkyl, heterocyclyalkyl, a        fluorescent moiety, a radioisotope, or a therapeutic agent;        R₇ and R₈ are independently —H, alkyl, alkenyl, alkynyl,        arylalkyl, cycloalkylalkyl, heteroalkylalkyl, or        heterocyclyalkyl;        R₉ and R₁₀ are each independently —H or any protecting group        suitable for liquid or solid phase peptide synthesis;

Q is S;

P is —H, -trityl, p-methoxytrityl, —S t-butyl, or any other protectinggroup suitable for liquid or solid phase peptide synthesis; or Q and Pwhen taken together form a moiety capable of undergoing chemicaltransformation into an —SH group; n is an integer from 1 to 5;andb) a macrocycle-forming linker of the structure:

X-L₂-Y

wherein L₂ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or[—R₁₁—K—R₁₁-]n, each being unsubstituted or substituted with R₁₂;each R₁₁ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;each R₁₂ is independently halogen, alkyl, —OR₁₃, —N(R₆)₁₃, —SR₁₃,—SOR₁₃, —SO₂R₁₃, —CO₂R₁₃, —R₁₃, a fluorescent moiety, a radioisotope, ora therapeutic agent;each R₁₃ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heteroalkylalkyl, heterocyclyalkyl, a fluorescentmoiety, a radioisotope, or a therapeutic agent; andX and Y are each independently a reactive group capable of reacting witha thiol group.

In some embodiments, the kit comprises one or more containers holdingone or more naturally-occurring amino acids or amino acid analogs asdescribed herein. In other embodiments, the kit comprises one or morecontainers holding one or more macrocycle-forming linkers as describedherein. In yet other embodiments, the kit comprises one or morecontainers holding one or more amino acids or amino acid analogs asdescribed herein, as well as one or more containers holding one or moremacrocycle-forming linkers as described herein.

For example, in some embodiments, the kit comprises a container holdingat least one amino acid or amino acid analog, as described above, havingan —SH moiety, the amino acid optionally protected and suitable for thesyntheses described herein. In some embodiments, the amino acid or aminoacid analog is selected from the group consisting of L-cysteine.D-cysteine, L-N-methylcysteine, D-N-methylcysteine, L-homocysteine,D-homocysteine, L-N-methylhomocysteine, D-N-methylhomocysteine,α-methyl-L-cysteine, α-methyl-D-cysteine, α-methyl-L-homocysteine,α-methyl-D-homocysteine, L-penicillamine, D-penicillamine,L-N-methylpenicillamine, D-N-methylpenicillamine and all forms suitablyprotected for liquid or solid phase peptide synthesis.

In some embodiments, the kit comprises a container holding at least onenaturally-occurring amino acid, non-naturally-occurring amino acid, oramino acid analog bound to a solid support compatible with the synthesesdescribed herein for peptidomimetic macrocycles.

In some embodiments, the kit comprises a container holding amacrocycle-forming linker as described above. In some embodiments, thekit further comprises one or more containers holding reagents necessaryfor the macrocyclization reactions described herein, such astrifluoracetic acid, liquid ammonia, NH₃/MeOH, NH₃/DMF, mercaptoethanol,hindered bases such as triethylamine or diisopropylethylamine, andguanidinium HCl.

In some embodiments, the kit comprises one container holding an aminoacid analog of the invention including a reactive —SH group incombination with a container holding a macrocycle-forming linker of theinvention.

Optionally, the kit further comprises one or more containers holdingreagents necessary for the macrocyclization reaction. In otherembodiments, the kit comprises two containers, each of which holds adifferent amino acid analog of the invention including a reactive —SHgroup. Optionally, the kit further comprises one or more containersholding reagents necessary for the macrocyclization reaction and/or amacrocycle-forming linker of the invention.

Assays

The properties of the peptidomimetic macrocycles of the invention areassayed, for example, by using the methods described below. In someembodiments, a macrocycle of the invention has enhanced propertiesrelative to a corresponding non-macrocyclic polypeptide. A correspondingnon-macrocyclic polypeptide is, for example, a precursor of apeptidomimetic macrocycle, such as a compound of Formula III which isconverted into said macrocycle. Alternatively, a correspondingnon-macrocyclic polypeptide is a polypeptide sequence, such as a naturalpolypeptide sequence which has substantial sequence overlap with themacrocycle of the invention. Numerous examples of natural polypeptidescorresponding to the macrocyclic polypeptide are shown in Tables 1, 2, 3and 4.

In general, a corresponding non-macrocyclic polypeptide can also be alabeled natural polypeptide or peptidomimetic precursor. Such labeling,for example by fluorescent or radioactive labeling, is used if necessaryin some of the assays described below. In such assays, both themacrocycle and the corresponding non-macrocyclic polypeptide aretypically labeled by similar or functionally equivalent methods.

Assay to Determine Alpha Helicity.

The percent helicity of unmodified pro-apoptotic BH3 domains arepredominantly random coils in solution, with α-helical content usuallyunder 25%. Peptidomimetic macrocycles with optimized linkers, on theother hand, possess, for example, an alpha-helicity that is at leasttwo-fold greater than that of a corresponding non-macrocyclicpolypeptide. In some embodiments, macrocycles of the invention willpossess an alpha-helicity of greater than 50%. To assay the helicity ofpeptidomimetic macrocycles of the invention, such as BH3 domain-basedmacrocycles, the compounds are dissolved in aqueous 50 mM potassiumphosphate solution at pH 7, or distilled H₂O, to concentrations of 25-50μM. Circular dichroism (CD) spectra are obtained on a spectropolarimeter(e.g., Jasco J-710) at 20° C. using the following standard measurementparameters: wavelength, 190-260 nm; step resolution, 0.5 nm; speed, 20nm/sec: accumulations, 10; response, 1 sec; bandwidth, 1 nm; pathlength, 0.1 cm. The α helical content of each peptide is calculated bydividing the mean residue ellipticity [Φ]222 obs by the reported [Φ]222obs for a model helical decapeptide (Yang et al. (1986), Methods Enymol.130:208)).

Assay to Determine Melting Temperature (Tm).

A peptidomimetic macrocycle of the invention comprising a secondarystructure such as an α-helix exhibits, for example, a higher meltingtemperature than a corresponding non-macrocyclic polypeptide. Typicallypeptidomimetic macrocycles of the invention exhibit Tm of >60° C.representing a highly stable structure in aqueous solutions.Peptidomimetic macrocycles and unmodified peptides are dissolved indistilled H₂O at final concentration of 50 μM and the Tm is determinedby measuring the change in ellipticity over a temperature range (4 to95° C.) on a spectropolarimeter (e.g., Jasco J-710) using the followingmeasurement parameters: wavelength 222 nm; step resolution, 0.5 nm;speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm;temperature increase rate: 1° C./min; path length, 0.1 cm.

Protease Resistance Assay.

The amide bond of the peptide backbone is susceptible to hydrolysis byproteases, thereby rendering peptidic compounds vulnerable to rapiddegradation in vivo. Peptide helix formation, however, typically buriesthe amide backbone and therefore may shield it from proteolyticcleavage. The peptidomimetic macrocycles of the present invention aresubjected to in vitro trypsin proteolysis to assess for any change indegradation rate compared to a corresponding non-macrocyclicpolypeptide. The peptidomimetic macrocycle and a correspondingnon-macrocyclic polypeptide are incubated with trypsin agarose and thereactions quenched at various time points by centrifugation andsubsequent HPLC injection to quantitate the residual substrate byultraviolet absorption at 280 nm. Briefly, the peptidomimetic macrocycleand precursor peptide (5 meg) are incubated with trypsin agarose(Pierce) (S/E ˜125) for 0, 10, 20, 90, and 180 minutes. Reactions arequenched by tabletop centrifugation at high speed; remaining substratein the isolated supernatant is quantified by HPLC-based peak detectionat 280 nm. The proteolytic reaction displays first order kinetics andthe rate constant, k, is determined from a plot of ln [S] versus time(k=−1×slope).

Ex Vivo Stability Assay.

Peptidomimetic macrocycles with optimized linkers possess, for example,an er vive half-life that is at least two-fold greater than that of acorresponding non-macrocyclic polypeptide peptide, and possess an exvivo half-life of 12 hours or more. For ex vivo serum stability studies,a peptidomimetic macrocycle and a corresponding non-macrocyclicpolypeptide (in a specific example, the corresponding naturalpolypeptide) (2 meg) are incubated with fresh mouse, rat and human serum(2 mL) at 37° C. for 0, 1, 2, 4, 8, and 24 hours. To determine the levelof intact compound, the following procedure is used: The samples areextracted by transferring 100 μl of sera to 2 ml centrifuge tubesfollowed by the addition of 10 μL of 50% formic acid and 500 μLacetonitrile and centrifugation at 14,000 RPM for 10 min at 4±2° C. Thesupernatants are then transferred to fresh 2 ml tubes and evaporated onTurbovap under N₂<10 psi, 37° C. The samples are reconstituted in 100 μLof 50:50 acetonitrile:water and submitted to LC-MS/MS analysis.

In Vitro Binding Assays.

To assess the binding and affinity of peptidomimetic macrocycles andprecursor peptides to acceptor proteins, a fluorescence polarizationassay (FPA) is used, for example. The FPA technique measures themolecular orientation and mobility using polarized light and fluorescenttracer. When excited with polarized light, fluorescent tracers (e.g.,FITC) attached to molecules with high apparent molecular weights (e.g.FITC-labeled peptides bound to a large protein) emit higher levels ofpolarized fluorescence due to their slower rates of rotation as comparedto fluorescent tracers attached to smaller molecules (e.g. FITC-labeledpeptides that are free in solution).

For example, fluoresceinated peptidomimetic macrocycles (25 nM) areincubated with the acceptor protein (25-1000 nM) in binding buffer (140mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature.Binding activity is measured, for example, by fluorescence polarizationon a Perkin-Elmer LS50B luminescence spectrophotonmeter. Kd values aredetermined by nonlinear regression analysis using Graphpad Prismsoftware. A peptidomimetic macrocycle of the invention show, forexample, similar or lower Kd than a corresponding non-macrocyclicpolypeptide.

Acceptor proteins for BH3-peptides such as BCL-2, BCL-X_(L), BAX or MCL1can be used in this assay. Acceptor proteins for p53 peptides such asMDM2 or MDMX can be used in this assay.

In Vitro Displacement Assays to Characterize Antagonists ofPeptide-Protein Interactions.

To assess the binding and affinity of compounds that antagonize theinteraction between a peptide (e.g. a BH3 peptide or a p53 peptide) andan acceptor protein, a fluorescence polarization assay (FPA) utilizing afluoresceinated peptidomimetic macrocycle derived from a precursorpeptide sequence is used, for example. The FPA technique measures themolecular orientation and mobility using polarized light and fluorescenttracer. When excited with polarized light, fluorescent tracers (e.g.,FITC) attached to molecules with high apparent molecular weights (e.g.FITC-labeled peptides bound to a large protein) emit higher levels ofpolarized fluorescence due to their slower rates of rotation as comparedto fluorescent tracers attached to smaller molecules (e.g. FITC-labeledpeptides that are free in solution). A compound that antagonizes theinteraction between the fluoresceinated peptidomimetic macrocycle and anacceptor protein will be detected in a competitive binding FPAexperiment.

For example, putative antagonist compounds (1 nM to 1 mM) and afluoresceinated peptidomimetic macrocycle (25 nM) are incubated with theacceptor protein (50 nM) in binding buffer (140 mM NaCl, 50 mM Tris-HCL,pH 7.4) for 30 minutes at room temperature. Antagonist binding activityis measured, for example, by fluorescence polarization on a Perkin-ElmerLS50B luminescence spectrophotometer. Kd values are determined bynonlinear regression analysis using Graphpad prism software.

Any class of molecule, such as small organic molecules, peptides,oligonucleotides or proteins can be examined as putative antagonists inthis assay. Acceptor proteins for BH3-peptides such as BCL2, BCL-XL. BAXor MCL1 can be used in this assay. Acceptor proteins for p53 peptidessuch as MDM2 or MDMX can be used in this assay.

Binding Assays in Intact Cells.

It is possible to measure binding of peptides or peptidomimeticmacrocycles to their natural acceptors in intact cells byimmunoprecipitation experiments. For example, intact cells are incubatedwith fluoresceinated (FITC-labeled) compounds for 4 hrs in the absenceof serum, followed by serum replacement and further incubation thatranges from 4-18 hrs. Cells are then pelleted and incubated in lysisbuffer (50 mM Tris [pH 7.6], 150 mM NaCl, 1% CHAPS and proteaseinhibitor cocktail) for 10 minutes at 4° C. Extracts are centrifuged at14.000 rpm for 15 minutes and supernatants collected and incubated with10 μl goat anti-FITC antibody for 2 hrs, rotating at 4° C. followed byfurther 2 hrs incubation at 4° C. with protein A/G Sepharose (50 μl of50% bead slurry). After quick centrifugation, the pellets are washed inlysis buffer containing increasing salt concentration (e.g., 150, 300,500 mM). The beads are then re-equilibrated at 150 mM NaCl beforeaddition of SDS-containing sample buffer and boiling. Aftercentrifugation, the supernatants are optionally electrophoresed using4%-12% gradient Bis-Tris gels followed by transfer into Immobilon-Pmembranes. After blocking, blots are optionally incubated with anantibody that detects FITC and also with one or more antibodies thatdetect proteins that bind to the peptidomimetic macrocycle, includingBCL2, MCL1, BCL-XL, Al, BAX, BAK, MDM2 or MDMX.

Cellular Permeability Assays.

A peptidomimetic macrocycle is, for example, more cell permeablecompared to a corresponding non-macrocyclic polypeptide. In someembodiments, the peptidomimetic macrocycles are more cell permeable thana corresponding non-macrocyclic polypeptides. Peptidomimetic macrocycleswith optimized linkers possess, for example, cell permeability that isat least two-fold greater than a corresponding non-macrocyclicpolypeptide, and often 20% or more of the applied peptide will beobserved to have penetrated the cell after 4 hours. To measure the cellpermeability of peptidomimetic macrocycles and correspondingnon-macrocyclic polypeptides, intact cells are incubated withfluoresceinated peptidomimetic macrocycles or correspondingnon-macrocyclic polypeptides (10 μM) for 4 hrs in serum free media at37° C. washed twice with media and incubated with trypsin (0.25%) for 10min at 37° C. The cells are washed again and resuspended in PBS.Cellular fluorescence is analyzed, for example, by using either aFACSCalibur flow cytometer or Cellomics' KineticScan® HCS Reader.

Cellular Efficacy Assays.

The efficacy of certain peptidomimetic macrocycles is determined, forexample, in cell-based killing assays using a variety of tumorigenic andnon-tumorigenic cell lines and primary cells derived from human or mousecell populations. Cell viability is monitored, for example, over 24-96hrs of incubation with peptidomimetic macrocycles (0.5 to 50 μM) toidentify those that kill at EC50<10 μM. Several standard assays thatmeasure cell viability are commercially available and are optionallyused to assess the efficacy of the peptidomimetic macrocycles. Inaddition, assays that measure Annexin V and caspase activation areoptionally used to assess whether the peptidomimetic macrocycles killcells by activating the apoptotic machinery.

In Vivo Stability Assay.

To investigate the in vivo stability of the peptidomimetic macrocycles,the compounds are, for example, administered to mice and/or rat by IV,IP, PO or inhalation routes at concentrations ranging from 0.1 to 50mg/Kg and blood specimens withdrawn at 0′, 5′, 15′, 30′, 1 hr, 4 hrs, 8hrs and 24 hours post-injection. Levels of intact compound in 25 L offresh serum are then measured by LC-MS/MS as above.

In Vivo Efficacy in Animal Models.

To determine the anti-oncogenic activity of the certain peptidomimeticmacrocycles in vivo, the compounds are, for example, given alone (IP,IV, PO, by inhalation or nasal routes) or in combination withsub-optimal doses of relevant chemotherapy (e.g., cyclophosphamide,doxorubicin, etoposide). In one example, 5×10⁶ RS4; 11 cells(established from the bone marrow of a patient with acute lymphoblasticleukemia) that stably express luciferase are injected by tail vein inNOD-SCID mice 3 hrs after they have been subjected to total bodyirradiation. If left untreated, this form of leukemia is fatal in 3weeks in this model. The leukemia is readily monitored, for example, byinjecting the mice with D-luciferin (60 mg/kg) and imaging theanesthetized animals (e.g., Xenogen In Vivo Imaging System, Caliper LifeSciences, Hopkinton, Mass.). Total body bioluminescence is quantified byintegration of photonic flux (photons/sec) by Living Image Software(Caliper Life Sciences, Hopkinton, Mass.). Peptidomimetic macrocyclesalone or in combination with sub-optimal doses of relevantchemotherapeutics agents are, for example, administered to leukemic mice(10 days after injection/day 1 of experiment, in bioluminescence of14-16) by tail vein or IP routes at doses ranging from 0.1 mg/kg to 50mg/kg for 7 to 21 days. Optionally, the mice are imaged throughout theexperiment every other day and survival monitored daily for the durationof the experiment. Expired mice are optionally subjected to necropsy atthe end of the experiment. Another animal model is implantation ofDoHH2, a cell line derived from human follicular lymphoma, into NOD-SCIDmice that stably expresses luciferase. These in vive tests optionallygenerate preliminary pharmacokinetic, pharmacodynamic and toxicologydata.

Clinical Trials.

To determine the suitability of the peptidomimetic macrocycles of theinvention for treatment of humans, clinical trials are performed. Forexample, patients diagnosed with cancer and in need of treatment areselected and separated in treatment and one or more control groups,wherein the treatment group is administered a peptidomimetic macrocycleof the invention, while the control groups receive a placebo or a knownanti-cancer drug. The treatment safety and efficacy of thepeptidomimetic macrocycles of the invention can thus be evaluated byperforming comparisons of the patient groups with respect to factorssuch as survival and quality-of-life. In this example, the patient grouptreated with a peptidomimetic macrocycle show improved long-termsurvival compared to a patient control group treated with a placebo.

Pharmaceutical Compositions and Routes of Administration

The peptidomimetic macrocycles of the invention also includepharmaceutically acceptable derivatives or prodrugs thereof. A“pharmaceutically acceptable derivative” means any pharmaceuticallyacceptable salt, ester, salt of an ester, pro-drug or other derivativeof a compound of this invention which, upon administration to arecipient, is capable of providing (directly or indirectly) a compoundof this invention. Particularly favored pharmaceutically acceptablederivatives are those that increase the bioavailability of the compoundsof the invention when administered to a mammal (e.g., by increasingabsorption into the blood of an orally administered compound) or whichincreases delivery of the active compound to a biological compartment(e.g., the brain or lymphatic system) relative to the parent species.Some pharmaceutically acceptable derivatives include a chemical groupwhich increases aqueous solubility or active transport across thegastrointestinal mucosa.

In some embodiments, the peptidomimetic macrocycles of the invention aremodified by covalently or non-covalently joining appropriate functionalgroups to enhance selective biological properties. Such modificationsinclude those which increase biological penetration into a givenbiological compartment (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism, and alter rate ofexcretion.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, benzoate, benzenesulfonate, butyrate, citrate,digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate,heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate,nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate,salicylate, succinate, sulfate, tartrate, tosylate and undecanoate.Salts derived from appropriate bases include alkali metal (e.g.,sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)₄⁻ salts.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers include eithersolid or liquid carriers. Solid form preparations include powders,tablets, pills, capsules, cachets, suppositories, and dispersiblegranules. A solid carrier can be one or more substances, which also actsas diluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material. Details ontechniques for formulation and administration are well described in thescientific and patent literature, see, e.g., the latest edition ofRemington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired.

Suitable solid excipients are carbohydrate or protein fillers include,but are not limited to sugars, including lactose, sucrose, mannitol, orsorbitol; starch from corn, wheat, rice, potato, or other plants;cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, orsodium carboxytnethylcellulose; and gums including arabic andtragacanth; as well as proteins such as gelatin and collagen. Ifdesired, disintegrating or solubilizing agents are added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

When the compositions of this invention comprise a combination of apeptidomimetic macrocycle and one or more additional therapeutic orprophylactic agents, both the compound and the additional agent shouldbe present at dosage levels of between about 1 to 100%, and morepreferably between about 5 to 95% of the dosage normally administered ina monotherapy regimen. In some embodiments, the additional agents areadministered separately, as part of a multiple dose regimen, from thecompounds of this invention. Alternatively, those agents are part of asingle dosage form, mixed together with the compounds of this inventionin a single composition.

Methods of Use

In one aspect, the present invention provides novel peptidomimeticmacrocycles that are useful in competitive binding assays to identifyagents which bind to the natural ligand(s) of the proteins or peptidesupon which the peptidomimetic macrocycles are modeled. For example, inthe p53 MDM2 system, labeled stabilized peptidomimetic macrocycles basedon the p53 is used in an MDM2 binding assay along with small moleculesthat competitively bind to MDM2. Competitive binding studies allow forrapid in vitro evaluation and determination of drug candidates specificfor the p53/MDM2 system. Likewise in the BH3/BCL-X_(L) anti-apoptoticsystem labeled peptidomimetic macrocycles based on BH3 can be used in aBCL-X_(L) binding assay along with small molecules that competitivelybind to BCL-X_(L). Competitive binding studies allow for rapid in vitroevaluation and determination of drug candidates specific for theBH3/BCL-X_(L) system. The invention further provides for the generationof antibodies against the peptidomimetic macrocycles. In someembodiments, these antibodies specifically bind both the peptidomimeticmacrocycle and the p53 or BH3 precursor peptides upon which thepeptidomimetic macrocycles are derived. Such antibodies, for example,disrupt the p53/MDM2 or BH3/BCL-XL systems, respectively.

In other aspects, the present invention provides for both prophylacticand therapeutic methods of treating a subject at risk of (or susceptibleto) a disorder or having a disorder associated with aberrant (e.g.,insufficient or excessive) BCL-2 family member expression or activity(e.g., extrinsic or intrinsic apoptotic pathway abnormalities). It isbelieved that some BCL-2 type disorders are caused, at least in part, byan abnormal level of one or more BCL-2 family members (e.g., over orunder expression), or by the presence of one or more BCL-2 familymembers exhibiting abnormal activity. As such, the reduction in thelevel and/or activity of the BCL-2 family member or the enhancement ofthe level and/or activity of the BCL-2 family member, is used, forexample, to ameliorate or reduce the adverse symptoms of the disorder.

In another aspect, the present invention provides methods for treatingor preventing hyperproliferative disease by interfering with theinteraction or binding between p53 and MDM2 in tumor cells. Thesemethods comprise administering an effective amount of a compound of theinvention to a warm blooded animal, including a human, or to tumor cellscontaining wild type p53. In some embodiments, the administration of thecompounds of the present invention induce cell growth arrest orapoptosis. In other or further embodiments, the present invention isused to treat disease and/or tumor cells comprising elevated MDM2levels. Elevated levels of MDM2 as used herein refers to MDM2 levelsgreater than those found in cells containing more than the normal copynumber (2) of mdm2 or above about 10,000 molecules of MDM2 per cell asmeasured by ELISA and similar assays (Picksley et al. (1994), Oncogene9, 2523 2529).

As used herein, the term “treatment” is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease or the predisposition toward disease.

In some embodiments, he peptidomimetics macrocycles of the invention isused to treat, prevent, and/or diagnose cancers and neoplasticconditions. As used herein, the temls “cancer”, “hyperproliferative” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyperproliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. A metastatic tumor can arise from a multitude of primarytumor types, including but not limited to those of breast, lung, liver,colon and ovarian origin. “Pathologic hyperproliferative” cells occur indisease states characterized by malignant tumor growth. Examples ofnon-pathologic hyperproliferative cells include proliferation of cellsassociated with wound repair. Examples of cellular proliferative and/ordifferentiative disorders include cancer, e.g., carcinoma, sarcoma, ormetastatic disorders. In some embodiments, the peptidomimeticsmacrocycles are novel therapeutic agents for controlling breast cancer,ovarian cancer, colon cancer, lung cancer, metastasis of such cancersand the like.

Examples of cancers or neoplastic conditions include, but are notlimited to, a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer,esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer,prostate cancer, uterine cancer, cancer of the head and neck, skincancer, brain cancer, squamous cell carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma choriocarcinoma, seminoma,embryonal carcinoma, Wilm's tumor, cervical cancer, testicular cancer,small cell lung carcinoma, non-small cell lung carcinoma, bladdercarcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma, leukemia, lymphoma, or Kaposi sarcoma.

Examples of proliferative disorders include hematopoietic neoplasticdisorders. As used herein, the term “hematopoietic neoplastic disorders”includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. Preferably, the diseases arisefrom poorly differentiated acute leukemias. e.g., erythroblasticleukemia and acute megakaryoblastic leukemia. Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus (1991), Crit Rev.Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereoft peripheral T cell lymphomas, adult T cell leukemia %lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granularlymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stemberg disease.

Examples of cellular proliferative and/or differentiative disorders ofthe breast include, but are not limited to, proliferative breast diseaseincluding, e.g., epithelial hyperplasia, sclerosing adenosis, and smallduct papillomas; tumors, e.g., stromal tumors such as fibroadenoma,phyllodes tumor, and sarcomas, and epithelial tumors such as large ductpapilloma; carcinoma of the breast including in situ (noninvasive)carcinoma that includes ductal carcinoma in situ (including Paget'sdisease) and lobular carcinoma in situ, and invasive (infiltrating)carcinoma including, but not limited to, invasive ductal carcinoma,invasive lobular carcinoma, medullary carcinoma, colloid (mucinous)carcinoma, tubular carcinoma, and invasive papillary carcinoma, andmiscellaneous malignant neoplasms. Disorders in the male breast include,but are not limited to, gynecomastia and carcinoma.

Examples of cellular proliferative and/or differentiative disorders ofthe lung include, but are not limited to, bronchogenic carcinoma,including paraneoplastic syndromes, bronchioloalveolar carcinoma,neuroendocrine tumors, such as bronchial carcinoid, miscellaneoustumors, and metastatic tumors; pathologies of the pleura, includinginflammatory pleural effusions, noninflammatory pleural effusions,pneumothorax, and pleural tumors, including solitary fibrous tumors(pleural fibroma) and malignant mesothelioma.

Examples of cellular proliferative and/or differentiative disorders ofthe colon include, but are not limited to, non-neoplastic polyps,adenomas, familial syndromes, colorectal carcinogenesis, colorectalcarcinoma, and carcinoid tumors.

Examples of cellular proliferative and/or differentiative disorders ofthe liver include, but are not limited to, nodular hyperplasias,adenomas, and malignant tumors, including primary carcinoma of the liverand metastatic tumors.

Examples of cellular proliferative and/or differentiative disorders ofthe ovary include, but are not limited to, ovarian tumors such as,tumors of coelomic epithelium, serous tumors, mucinous tumors,endometrioid tumors, clear cell adenocarcinoma, cystadenofibroma,Brenner tumor, surface epithelial tumors; germ cell tumors such asmature (benign) teratomas, monodermal teratomas, immature malignantteratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sexcord-stomal tumors such as, granulosa-theca cell tumors,thecomafibromas, androblastomas, hill cell tumors, and gonadoblastoma;and metastatic tumors such as Krukenberg tumors.

In other or further embodiments, the peptidomimetics macrocyclesdescribed herein are used to treat, prevent or diagnose conditionscharacterized by overactive cell death or cellular death due tophysiologic insult, etc. Some examples of conditions characterized bypremature or unwanted cell death are or alternatively unwanted orexcessive cellular proliferation include, but are not limited tohypocellular/hypoplastic, acellular/aplastic, orhypercellular/hyperplastic conditions. Some examples include hematologicdisorders including but not limited to fanconi anemia, aplastic anemia,thalaessemia, congenital neutropenia, myelodysplasia

In other or further embodiments, the peptidomimetics macrocycles of theinvention that act to decrease apoptosis are used to treat disordersassociated with an undesirable level of cell death. Thus, in someembodiments, the anti-apoptotic peptidomimetics macrocycles of theinvention are used to treat disorders such as those that lead to celldeath associated with viral infection, e.g., infection associated withinfection with human immunodeficiency virus (HIV). A wide variety ofneurological diseases are characterized by the gradual loss of specificsets of neurons, and the anti-apoptotic peptidomimetics macrocycles ofthe invention are used, in some embodiments, in the treatment of thesedisorders. Such disorders include Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis (ALS) retinitis pigmentosa,spinal muscular atrophy, and various forms of cerebellar degeneration.The cell loss in these diseases does not induce an inflammatoryresponse, and apoptosis appears to be the mechanism of cell death. Inaddition, a number of hematologic diseases are associated with adecreased production of blood cells. These disorders include anemiaassociated with chronic disease, aplastic anemia, chronic neutropenia,and the myelodysplastic syndromes. Disorders of blood cell production,such as myelodysplastic syndrome and some forms of aplastic anemia, areassociated with increased apoptotic cell death within the bone marrow.These disorders could result from the activation of genes that promoteapoptosis, acquired deficiencies in stromal cells or hematopoieticsurvival factors, or the direct effects of toxins and mediators ofimmune responses. Two common disorders associated with cell death aremyocardial infarctions and stroke. In both disorders, cells within thecentral area of ischemia, which is produced in the event of acute lossof blood flow, appear to die rapidly as a result of necrosis. However,outside the central ischemic zone, cells die over a more protracted timeperiod and morphologically appear to die by apoptosis. In other orfurther embodiments, the anti-apoptotic peptidomimetics macrocycles ofthe invention are used to treat all such disorders associated withundesirable cell death.

Some examples of immunologic disorders that are treated with thepeptidomimetics macrocycles described herein include but are not limitedto organ transplant rejection, arthritis, lupus, IBD, Crohn's disease,asthma, multiple sclerosis, diabetes, etc.

Some examples of neurologic disorders that are treated with thepeptidomimetics macrocycles described herein include but are not limitedto Alzheimer's Disease, Down's Syndrome, Dutch Type Hereditary CerebralHemorrhage Amyloidosis. Reactive Amyloidosis, Familial AmyloidNephropathy with Urticaria and Deafness, Muckle-Wells Syndrome,Idiopathic Myeloma; Macroglobulinemia-Associated Myeloma, FamilialAmyloid Polyneuropathy, Familial Amyloid Cardiomyopathy. IsolatedCardiac Amyloid, Systemic Senile Amyloidosis, Adult Onset Diabetes,Insulinoma, Isolated Atrial Amyloid, Medullary Carcinoma of the Thyroid,Familial Amyloidosis. Hereditary Cerebral Hemorrhage With Amyloidosis,Familial Amyloidotic Polyneuropathy, Scrapie, Creutzfeldt-Jacob Disease,Gerstmann Straussler-Scheinker Syndrome, Bovine Spongiform Encephalitis,a prion-mediated disease, and Huntington's Disease.

Some examples of endocrinologic disorders that are treated with thepeptidomimetics macrocycles described herein include but are not limitedto diabetes, hypothyroidism, hypopituitarism, hypoparathyroidism,hypogonadism, etc.

Examples of cardiovascular disorders (e.g., inflammatory disorders) thatare treated or prevented with the peptidomimetics macrocycles of theinvention include, but are not limited to, atherosclerosis, myocardialinfarction, stroke, thrombosis, aneurism, heart failure, ischemic heartdisease, angina pectoris, sudden cardiac death, hypertensive heartdisease; non-coronary vessel disease, such as arteriolosclerosis, smallvessel disease, nephropathy, hypertriglyceridemia, hypercholesterolemia,hyperlipidemia, xanthomatosis, asthma, hypertension, emphysema andchronic pulmonary disease; or a cardiovascular condition associated withinterventional procedures (“procedural vascular trauma”), such asrestenosis following angioplasty, placement of a shunt, stent, syntheticor natural excision grafts, indwelling catheter, valve or otherimplantable devices. Preferred cardiovascular disorders includeatherosclerosis, myocardial infarction, aneurism, and stroke.

EXAMPLES

The following section provides illustrative examples of the presentinvention.

Example 1. Synthesis of a Peptidomimetic Macrocycle

The target molecule is the BID-BH3 peptide with amino acids 12 and 16replaced by Cysteine (see Table 1 and Table 6). The Cysteine side chainthiols are then derivatized with 1,4-dibromobutane to form thebis-thioether peptidomimetic macrocycle.

Following the general peptide synthetic strategy described, thepeptidomimetic precursor was a polypeptide of the sequenceDIIARHLACVGDCN_(L)DRSI (where “N_(L)” or “Nle” represent norleucine)synthesized at 0.2 mmol scale on a PTI-Ranin PS3 single channelsynthesizer using the following coupling cycles for each amino acid:

Deprotection 20% piperidine in DMF 2 × 7 min Wash DMF 6 × 0.5 minCoupling 5 fold excess each of amino acid, TBTU and DIEA in DMF 1 × 20min Wash DMF 2 × 0.5 min Coupling 5 fold excess each of amino acid, TBTUand DIEA in DMF 1 × 20 min Wash DMF 6 × 0.5 min

The precursor polypeptide was acetylated at the amino terminus bytreatment with 1 mM acetic anhydride and 1 mM diisopropylethylamine(DIEA) in dimethylformamide (DMF) for 45 minutes. Synthesis was done onrink amide resin (substitution 0.6 mMol/g) with the cys⁹ and cys¹³thiols protected with p-methoxytrityl (Mmt) groups. The Mmt groups wereselectively deprotected with 1% TFA/4% TIS in dichloromethane (DCM) andthe polypeptide was alkylated overnight at room temperature using 50molar equivalents of 1,4-dibromobutane and 13 molar equivalents ofdiisopropylethylamine (DIEA) in dichloroethane. The peptide was thencleaved from the resin by treatment with 94% TFA 2% TIS 2% Anisole 2%H₂O for 3 hours followed by filtration, concentration by rotaryevaporation and precipitation with diethyl ether. The expected molecularweight of the final peptidomimetic macrocycle product is 2448.91. Theobserved molecular weight is 2445.5 by MALDI MS (see FIG. 1).

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1-42. (canceled)
 43. A method of inhibiting MDMX or MDM2 activity in a subject, the method comprising administering to the subject a peptidomimetic macrocycle of Formula (I):

wherein: each A, C, D, and E is independently a natural or non-natural amino acid; each B is independently a natural or non-natural amino acid, amino acid analog,

 [—NH-L₄-CO—], [—NH-L₄-SO₂—], or [—NH-L₄-]; R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; each R₃ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, unsubstituted or substituted with R₅; L₁, L₃ and L₄ are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene or [—R₄—K—R₄-]n, each being unsubstituted or substituted with R₅; L₂ is alkylene, alkenylene, alkynylene, heteroalkylene, heterocycloalkylene, or [—R₄—K—R₄-]n, each being unsubstituted or substituted with R₅; each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃; each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent; each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, unsubstituted or substituted with R₅, or part of a cyclic structure with a D residue; R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, unsubstituted or substituted with R₅, or part of a cyclic structure with an E residue; v is an integer from 1-1000; w is an integer from 1-1000; x is an integer from 0-10; y is an integer from 0-10; z is an integer from 0-10; and n is an integer from 1-5.
 44. The method of claim 43, wherein at least one of R₁ and R₂ is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-.
 45. The method of claim 43, wherein at least one of R₁ and R₂ is alkyl, unsubstituted or substituted with halo-.
 46. The method of claim 43, wherein at least one of D and E is attached to an additional macrocycle-forming linker of the formula [-L₁-S-L₂-S-L₃-].
 47. The method of claim 43, wherein a secondary structure of the peptidomimetic macrocycle is more stable than a corresponding secondary structure of a corresponding non-macrocyclic polypeptide.
 48. The method of claim 43, wherein the peptidomimetic macrocycle comprises an α-helix.
 49. The method of claim 43, wherein [-L₁-S-L₂-S-L₃-] spans approximately 1, 2, 3, 4 or 5 turns of the α-helix.
 50. The method of claim 48, wherein the length of [-L₁-S-L₂-S-L₃-] is about 5 Å to about 9 Å per turn of the α-helix.
 51. The method of claim 48, wherein the length of [-L₁-S-L₂-S-L₃-] is approximately equal to the length of from about 5 carbon-carbon bonds to about 13 carbon-carbon bonds.
 52. The method of claim 48, wherein the macrocycle comprises a ring of about 17 atoms to 25 atoms or a ring of about 29 atoms to about 37 atoms.
 53. The method of claim 43, wherein x+y+z is at least
 3. 54. The method of claim 43, wherein L₂ is [R₄—K—R₄-]n.
 55. The method of claim 43, wherein R₄ is arylene.
 56. The method of claim 43, wherein K is CO.
 57. The method of claim 43, wherein R₄ is phenylene and n is
 1. 58. The method of claim 57, wherein L₁ and L₃ are alkylene.
 59. The method of claim 57, wherein L₁ and L₃ are methylene.
 60. The method of claim 57, wherein the subject is a human.
 61. The method of claim 43, wherein the subject suffers from a hyperproliferative disease.
 62. The method of claim 61, wherein the hyperproliferative disease is a cancer.
 63. The method of claim 43, wherein the subject suffers from a neoplastic disease. 